Placenta-derived adherent cell exosomes and uses thereof

ABSTRACT

Provided herein are compositions of placenta-derived adherent cell exosomes and methods of making and using the same. In one aspect, provided herein are compositions comprising exosomes produced by and/or derived from placental cells, e.g., placenta-derived adherent cells. In certain embodiments, the exosomes provided herein are produced by placenta-derived adherent cells that have been cultured in vitro for, e.g., 1, 2, 3, 4, 5, 6 or more passages.

This application claims benefit of U.S. Provisional Patent ApplicationNo. 62/062,046, filed Oct. 9, 2014, the disclosure of which isincorporated by reference herein in its entirety.

1. FIELD

The disclosure herein relates to compositions of placenta-derivedadherent cell exosomes and methods of making and using the same.

2. BACKGROUND

Exosomes are small, non-cellular bodies derived from living cells.Generally, exosomes are 50-150 nanometers (nm) in diameter and composedof a phospholipid bilayer derived from multivesicular bodies or theplasma membrane of eukaryotic cells. Exosomes may be round in shape butalso can be “cup-shaped” bodies that are visually identifiable usingelectron microscopy and other imaging techniques. Exosomes do notcontain an intact nucleus or the requisite cellular components tosupport the metabolic and/or molecular functions of a cell, but exosomesmay contain proteins, nucleic acids, and other molecules, or can bedecorated with surface proteins, which can aid in their detection andisolation. The content of exosomes can reflect the cell-type from whichthey are derived and can consist of molecular markers that reflect thefunctional state of the cells of origin. Exosomes hold promise asresearch tools and as a therapeutic modality, but there is a need in theart for new and useful exosomes.

3. SUMMARY

In one aspect, provided herein are compositions comprising exosomesproduced by and/or derived from placental cells, e.g., placenta-derivedadherent cells. In certain embodiments, the exosomes provided herein areproduced by placenta-derived adherent cells that have been cultured invitro for, e.g., 1, 2, 3, 4, 5, 6 or more passages. In certainembodiments, the exosomes provided herein are collected fromplacenta-derived adherent cells that have been cultured in vitro for 1passage. In other certain embodiment, the exosomes provided herein arecollected from placenta-derived adherent cells that have been culturedin vitro for 2 passages. In other certain embodiment, the exosomesprovided herein are collected from placenta-derived adherent cells thathave been cultured in vitro for 3 passages. In other certain embodiment,the exosomes provided herein are collected from placenta-derivedadherent cells that have been cultured in vitro for 4 passages. In othercertain embodiment, the exosomes provided herein are collected fromplacenta-derived adherent cells that have been cultured in vitro for 5passages. In a specific embodiment, the exosomes provided herein arecollected from placenta-derived adherent cells that have been culturedin vitro for 6 passages. In other certain embodiment, the exosomesprovided herein are collected from placenta-derived adherent cells thathave been cultured in vitro for more than 6 passages. In certainembodiments, the exosomes provided herein are collected fromplacenta-derived adherent cells plated at passage 6, and collected(e.g., harvested) from culture supernatant at passage 7.

The placenta-derived adherent cell exosomes described herein compriseparticular markers. Such markers can, for example, be useful in theidentification of the exosomes and for distinguishing them from otherexosomes, e.g., exosomes not derived from placenta-derived adherentcells. See Section 5.1.1. In certain embodiments, the placenta-derivedadherent cell exosomes provided herein comprise one, two, three, four,or more of CD9, CD10, CD13, CD29, CD44, CD49b, CD49c, CD55, CD59, CD63,CD73, CD81, CD82, CD90, CD98, CD105, CD141, CD142, CD151, CD164, CD295,and/or CD200, i.e., such exosomes are one or more of CD9⁺, CD10⁺, CD13⁺,CD29⁺, CD44⁺, CD49b⁺, CD49c⁺, CD55⁺, CD59⁺, CD63⁺, CD73⁺, CD81⁺, CD82⁺,CD90⁺, CD98⁺, CD105⁺, CD141⁺, CD142⁺, CD151⁺, CD164⁺, CD295⁺, and/orCD200⁺, e.g., as determinable by flow cytometry, for example, byfluorescence-activated cell sorting (FACS). In addition, theplacenta-derived adherent cell exosomes provided herein can beidentified based on the absence of certain markers. In certainembodiments, the placenta-derived adherent cell exosomes provided hereinlack one, two, three, four, or more of the following markers: CD3,CD11b, CD14, CD19, CD33, CD192, HLA-A, HLA-B, HLA-C, HLA-DR, CD11cand/or CD34, i.e., such exosomes are one or more of CD3⁻, CD11b⁻, CD14⁻,CD19⁻, CD33⁻, CD192⁻, HLA-A⁻, HLA-B⁻, HLA-C, HLA-DR⁻, CD11c⁻ and/orCD34⁻, e.g., as determinable by flow cytometry, for example, by FACS.Determination of the presence or absence of such markers can beaccomplished using methods known in the art, e.g.,fluorescence-activated cell sorting (FACS).

In a specific embodiment, the placenta-derived adherent cell exosomesdescribed herein are CD55⁺ and CD10⁺ as determined, e.g., by flowcytometry, for example, by FACS.

In specific embodiments, the placenta-derived adherent cell exosomesdescribed herein are CD10⁺ and CD55⁺ (as determined by, e.g., FACS) andadditionally contain CD82, CD142, CD49c, and CD90 at a level higher thanchorionic villi mesenchymal stem cell exosomes or pre-adipocytemesenchymal stem cell exosomes (e.g., the chorionic villi mesenchymalstem cells described in Salomon et al., 2013, PLOS ONE, 8:7, e68451).

In another embodiment, the placenta-derived adherent cell exosomesdescribed herein do not contain detectable levels of CD4, CD5, CD6, CD7,CD8, CD16, CD24, CD25, CD26, CD27, CD32, CD35, CD37, CD39, CD45, CD46,CD54, CD56, CD61, CD74, CD86, CD88, CD91, CD99, CD103, CD108, CD112,CD117, CD119, CD120a, CD123, CD126, CD130, CD134, CD138, CD140a, CD140b,CD144, CD146, CD147, CD152, CD163, CD183, CD184, CD186, CD191, CD194,CD196, CDw198, CD202b, CD220, CD221, CD235a, CD252, CD266, CD271, CD273,CD274, CD318, CD326, CD333, CD334, HLA-DP, HLA-DQ, HLA-G, IgD, IgM, NG2,PDGF, and/or SSEA-3, as assessed, e.g., by flow cytometry, for example,by FACS. In certain embodiments, the placenta-derived adherent cellexosomes do not contain detectable levels of CD4. In certainembodiments, the placenta-derived adherent cell exosomes do not containdetectable levels of CD5. In certain embodiments, the placenta-derivedadherent cell exosomes do not contain detectable levels of CD6. Incertain embodiments, the placenta-derived adherent cell exosomes do notcontain detectable levels of CD7. In certain embodiments, theplacenta-derived adherent cell exosomes do not contain detectable levelsof CD8. In certain embodiments, the placenta-derived adherent cellexosomes do not contain detectable levels of CD16. In certainembodiments, the placenta-derived adherent cell exosomes do not containdetectable levels of CD24. In certain embodiments, the placenta-derivedadherent cell exosomes do not contain detectable levels of CD25. Incertain embodiments, the placenta-derived adherent cell exosomes do notcontain detectable levels of CD26. In certain embodiments, theplacenta-derived adherent cell exosomes do not contain detectable levelsof CD27. In certain embodiments, the placenta-derived adherent cellexosomes do not contain detectable levels of CD32. In certainembodiments, the placenta-derived adherent cell exosomes do not containdetectable levels of CD35. In certain embodiments, the placenta-derivedadherent cell exosomes do not contain detectable levels of CD37. Incertain embodiments, the placenta-derived adherent cell exosomes do notcontain detectable levels of CD39. In certain embodiments, theplacenta-derived adherent cell exosomes do not contain detectable levelsof CD45. In certain embodiments, the placenta-derived adherent cellexosomes do not contain detectable levels of CD46. In certainembodiments, the placenta-derived adherent cell exosomes do not containdetectable levels of CD54. In certain embodiments, the placenta-derivedadherent cell exosomes do not contain detectable levels of CD56. Incertain embodiments, the placenta-derived adherent cell exosomes do notcontain detectable levels of CD61. In certain embodiments, theplacenta-derived adherent cell exosomes do not contain detectable levelsof CD74. In certain embodiments, the placenta-derived adherent cellexosomes do not contain detectable levels of CD86. In certainembodiments, the placenta-derived adherent cell exosomes do not containdetectable levels of CD88. In certain embodiments, the placenta-derivedadherent cell exosomes do not contain detectable levels of CD91. Incertain embodiments, the placenta-derived adherent cell exosomes do notcontain detectable levels of CD99. In certain embodiments, theplacenta-derived adherent cell exosomes do not contain detectable levelsof CD103. In certain embodiments, the placenta-derived adherent cellexosomes do not contain detectable levels of CD108. In certainembodiments, the placenta-derived adherent cell exosomes do not containdetectable levels of CD112. In certain embodiments, the placenta-derivedadherent cell exosomes do not contain detectable levels of CD117. Incertain embodiments, the placenta-derived adherent cell exosomes do notcontain detectable levels of CD119, In certain embodiments, theplacenta-derived adherent cell exosomes do not contain detectable levelsof CD120a. In certain embodiments, the placenta-derived adherent cellexosomes do not contain detectable levels of CD123. In certainembodiments, the placenta-derived adherent cell exosomes do not containdetectable levels of CD126. In certain embodiments, the placenta-derivedadherent cell exosomes do not contain detectable levels of CD130. Incertain embodiments, the placenta-derived adherent cell exosomes do notcontain detectable levels of CD134. In certain embodiments, theplacenta-derived adherent cell exosomes do not contain detectable levelsof CD138. In certain embodiments, the placenta-derived adherent cellexosomes do not contain detectable levels of CD140a. In certainembodiments, the placenta-derived adherent cell exosomes do not containdetectable levels of CD140b. In certain embodiments, theplacenta-derived adherent cell exosomes do not contain detectable levelsof CD144. In certain embodiments, the placenta-derived adherent cellexosomes do not contain detectable levels of CD146. In certainembodiments, the placenta-derived adherent cell exosomes do not containdetectable levels of CD147. In certain embodiments, the placenta-derivedadherent cell exosomes do not contain detectable levels of CD152. Incertain embodiments, the placenta-derived adherent cell exosomes do notcontain detectable levels of CD163. In certain embodiments, theplacenta-derived adherent cell exosomes do not contain detectable levelsof CD183. In certain embodiments, the placenta-derived adherent cellexosomes do not contain detectable levels of CD184. In certainembodiments, the placenta-derived adherent cell exosomes do not containdetectable levels of CD186. In certain embodiments, the placenta-derivedadherent cell exosomes do not contain detectable levels of CD191. Incertain embodiments, the placenta-derived adherent cell exosomes do notcontain detectable levels of CD194. In certain embodiments, theplacenta-derived adherent cell exosomes do not contain detectable levelsof CD196. In certain embodiments, the placenta-derived adherent cellexosomes do not contain detectable levels of CDw198. In certainembodiments, the placenta-derived adherent cell exosomes do not containdetectable levels of CD202b. In certain embodiments, theplacenta-derived adherent cell exosomes do not contain detectable levelsof CD220. In certain embodiments, the placenta-derived adherent cellexosomes do not contain detectable levels of CD221. In certainembodiments, the placenta-derived adherent cell exosomes do not containdetectable levels of CD235a. In certain embodiments, theplacenta-derived adherent cell exosomes do not contain detectable levelsof CD252. In certain embodiments, the placenta-derived adherent cellexosomes do not contain detectable levels of CD266. In certainembodiments, the placenta-derived adherent cell exosomes do not containdetectable levels of CD271. In certain embodiments, the placenta-derivedadherent cell exosomes do not contain detectable levels of CD273. Incertain embodiments, the placenta-derived adherent cell exosomes do notcontain detectable levels of CD274. In certain embodiments, theplacenta-derived adherent cell exosomes do not contain detectable levelsof CD318. In certain embodiments, the placenta-derived adherent cellexosomes do not contain detectable levels of CD326. In certainembodiments, the placenta-derived adherent cell exosomes do not containdetectable levels of CD333. In certain embodiments, the placenta-derivedadherent cell exosomes do not contain detectable levels of CD334. Incertain embodiments, the placenta-derived adherent cell exosomes do notcontain detectable levels of HLA-DP. In certain embodiments, theplacenta-derived adherent cell exosomes do not contain detectable levelsof HLA-DQ. In certain embodiments, the placenta-derived adherent cellexosomes do not contain detectable levels of HLA-G. In certainembodiments, the placenta-derived adherent cell exosomes do not containdetectable levels of IgD. In certain embodiments, the placenta-derivedadherent cell exosomes do not contain detectable levels of IgM. Incertain embodiments, the placenta-derived adherent cell exosomes do notcontain detectable levels of NG2. In certain embodiments, theplacenta-derived adherent cell exosomes do not contain detectable levelsof PDGF. In certain embodiments, the placenta-derived adherent cellexosomes do not contain detectable levels of SSEA-3.

In certain embodiments, the placenta-derived adherent cell exosomesdescribed herein are CD82⁺ and contain a low level of CD141 (i.e., areCD141(low)) as determined, e.g., by flow cytometry, for example, byFACS.

In certain embodiments, the placenta-derived adherent cell exosomesdescribed herein comprise one or more markers at a level at leasttwo-fold higher than the level of the marker as present in an equivalentnumber, or an equivalent mass, of exosomes derived from chorionic villimesenchymal stem cells or pre-adipocyte mesenchymal stem cells asdeterminable by, e.g., FACS. In a specific embodiment, theplacenta-derived adherent cell exosomes described herein comprise CD49c,CD142, CD90, and/or CD82 at a level at least two-fold higher than thelevel of each marker, respectively, as present in exosomes derived fromchorionic villi mesenchymal stem cells or pre-adipocyte mesenchymal stemcells, e.g., as determinable by flow cytometry, for example, by FACS.

In a specific embodiment, the placenta-derived adherent cell exosomesdescribed herein comprise a higher amount of CD49c, CD82, CD90, and/orCD142 than the amount of said marker(s) in a non placenta-derivedadherent cell exosome. In a specific embodiment, the placenta-derivedadherent cell exosomes described herein comprise a higher amount ofCD49c, CD82, CD90, and/or CD142 than the amount of said marker(s) thatis present in exosomes derived from chorionic villi mesenchymal stemcells (e.g., the chorionic villi mesenchymal stem cells described inSalomon et al., 2013, PLOS ONE, 8:7, e68451). In a specific embodiment,the placenta-derived adherent cell exosomes described herein comprise ahigher amount of CD49c, CD73, CD82, CD90, and/or CD142 than the amountof said marker(s) that is present in exosomes derived from pre-adipocytemesenchymal stem cells.

In certain embodiments, the placenta-derived adherent cell exosomesdescribed herein comprise one or more markers at a level at leasttwo-fold lower than the level of the marker as present in an equivalentnumber, or an equivalent mass, of exosomes derived from chorionic villimesenchymal stem cells or pre-adipocyte mesenchymal stem cells asdeterminable by, e.g., FACS.

In a specific embodiment, the placenta-derived adherent cell exosomesdescribed herein comprise a lower amount of CD164 than the amount ofsaid marker(s) in a non placenta-derived adherent cell exosome. In aspecific embodiment, the placenta-derived adherent cell exosomesdescribed herein comprise a lower amount of CD164 than the amount ofsaid marker(s) that is present in exosomes derived from pre-adipocytemesenchymal stem cells or chorionic villi mesenchymal stem cells (e.g.,the chorionic villi mesenchymal stem cells described in Salomon et al.,2013, PLOS ONE, 8:7, e68451).

In certain embodiments, the placenta-derived adherent cell exosomesdescribed herein comprise one or more nucleic acids. In one embodiment,said nucleic acids are non-coding RNAs. In another embodiment, saidnon-coding RNAs are microRNAs (miRNAs). In a specific embodiment, theplacenta-derived adherent cell exosomes described herein comprise one ormore miRNAs selected from the group consisting of: miR-218-5p, miR-133b,miR-422a, miR-564, miR-16-5p, let-7a, miR-92, miR-142-3p, miR-451,miR-124-5p, miR-223-3p, miR-630, miR-296-5p, let-7b-3p and let-7d-3p. Ina certain embodiment, the placenta-derived adherent cell exosomescomprise an miRNA that is miR-218-5p. In a certain embodiment, theplacenta-derived adherent cell exosomes comprise an miRNA that ismiR-133b. In a certain embodiment, the placenta-derived adherent cellexosomes comprise an miRNA that is miR-422a. In a certain embodiment,the placenta-derived adherent cell exosomes comprise an miRNA that ismiR-564, miR-16-5p. In a certain embodiment, the placenta-derivedadherent cell exosomes comprise an miRNA that is let-7a. In a certainembodiment, the placenta-derived adherent cell exosomes comprise anmiRNA that is miR-92. In a certain embodiment, the placenta-derivedadherent cell exosomes comprise an miRNA that is miR-142-3p. In acertain embodiment, the placenta-derived adherent cell exosomes comprisean miRNA that is miR-451. In a certain embodiment, the placenta-derivedadherent cell exosomes comprise an miRNA that is miR-124-5p. In acertain embodiment, the placenta-derived adherent cell exosomes comprisean miRNA that is miR-223-3p. In a certain embodiment, theplacenta-derived adherent cell exosomes comprise an miRNA that ismiR-630. In a certain embodiment, the placenta-derived adherent cellexosomes comprise an miRNA that is miR-296-5p. In a certain embodiment,the placenta-derived adherent cell exosomes comprise an miRNA that islet-7b-3p. In a certain embodiment, the placenta-derived adherent cellexosomes comprise an miRNA that is let-7d-3p. In another specificembodiment, the placenta-derived adherent cell exosomes comprise one ormore of miRNAs miR-218-5p, miR-133b, miR-422a, and/or miR-564. Inanother specific embodiment, the placenta-derived adherent cell exosomescomprise one or more of miRNAs miR-133b, miR-422a, miR-16-5p, miR-92,miR-142-3p, miR-451, miR-223-3p, miR-296-5p, and/ormiR-Let-7d*/Let-7d-3p. In other specific embodiments, theplacenta-derived adherent cell exosomes comprise one or more of miRNAsmiR-591, miR-218-5p, miR-133b, miR-422a, and/or miR-564. In anotherspecific embodiment, said one or more miRNAs are present at a level atleast two-fold higher than the level of the corresponding miRNA aspresent in chorionic villi mesenchymal stem cells. In other specificembodiments, the placenta-derived adherent cell exosomes, when subjectedto RT-PCR with respect to a specific miRNA and compared to a control RNA(e.g., RNU44), are contained at a level 1.1, 2, 5, 10, 100, 500, 600,700, 800, 900, 1000, 1100 or 1200 times higher than the same miRNA inchorionic villi mesenchymal stem cell exosomes, or between 1.1 and 2, 2and 5, 2 and 10, 5 and 10, 10 and 100, 100 and 500, 500 and 600, 600 and700, 700 and 800, 800 and 900, 900 and 1000, 1000 and 1100, or 1100 and1200 times higher than the same miRNA in chorionic villi mesenchymalstem cell exosomes. In a certain embodiment, the placenta-derivedadherent cell exosomes comprise at least one marker at a level at least1.1 times higher than exosomes derived from mesenchymal stem cells. In acertain embodiment, the placenta-derived adherent cell exosomes compriseat least one marker at a level at least 2 times higher than exosomesderived from mesenchymal stem cells. In a certain embodiment, theplacenta-derived adherent cell exosomes comprise at least one marker ata level at least 5 times higher than exosomes derived from mesenchymalstem cells. In a certain embodiment, the placenta-derived adherent cellexosomes comprise at least one marker at a level at least 10 timeshigher than exosomes derived from mesenchymal stem cells. In a certainembodiment, the placenta-derived adherent cell exosomes comprise atleast one marker at a level at least 100 times higher than exosomesderived from mesenchymal stem cells. In a certain embodiment, theplacenta-derived adherent cell exosomes comprise at least one marker ata level at least 500 times higher than exosomes derived from mesenchymalstem cells. In a certain embodiment, the placenta-derived adherent cellexosomes comprise at least one marker at a level at least 600 timeshigher than exosomes derived from mesenchymal stem cells. In a certainembodiment, the placenta-derived adherent cell exosomes comprise atleast one marker at a level at least 700 times higher than exosomesderived from mesenchymal stem cells. In a certain embodiment, theplacenta-derived adherent cell exosomes comprise at least one marker ata level at least 800 times higher than exosomes derived from mesenchymalstem cells. In a certain embodiment, the placenta-derived adherent cellexosomes comprise at least one marker at a level at least 900 timeshigher than exosomes derived from mesenchymal stem cells. In a certainembodiment, the placenta-derived adherent cell exosomes comprise atleast one marker at a level at least 1000 times higher than exosomesderived from mesenchymal stem cells. In a certain embodiment, theplacenta-derived adherent cell exosomes comprise at least one marker ata level at least 1100 times higher than exosomes derived frommesenchymal stem cells. In a certain embodiment, the placenta-derivedadherent cell exosomes comprise at least one marker at a level at least1200 times higher than exosomes derived from mesenchymal stem cells.

In one embodiment, the placenta-derived adherent cell exosomes describedherein contain one or more angiogenic proteins selected from the groupof Endoglin, Leptin, Angiopoietin-2, G-CSF, Follistatin, FGF-2, HGF,VEGF-A, IL-8 and/or EGF.

Further provided herein are populations of exosomes comprising theplacenta-derived adherent cell exosomes described herein. In a specificembodiment, the populations of exosomes provided herein are pure orsubstantially pure with respect to their content of placenta-derivedadherent cell exosomes, e.g., the populations of exosomes comprise about90%, 95%, 98%, 99%, or 100% placenta-derived adherent cell exosomes. Inanother specific embodiment, the populations of exosomes provided hereincomprise the placenta-derived adherent cell exosomes provided herein andone or more other types of exosomes, e.g., exosomes derived from a cellother than a placenta-derived adherent cell.

In a specific embodiment, provided herein is a population ofplacenta-derived adherent cell exosomes, wherein about 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of theexosomes in said population are one or more of CD9⁺, CD10⁺, CD13⁺,CD29⁺, CD44⁺, CD49b⁺, CD49c′, CD55⁺, CD59⁺, CD63⁺, CD73⁺, CD81⁺, CD82⁺,CD90⁺, CD98⁺, CD105⁺, CD141⁺, CD142⁺, CD151⁺, CD164⁺, CD295⁺, and/orCD200⁺, as determinable by, e.g., FACS.

The placenta-derived adherent cell exosomes described herein may beisolated by any method known in the art suitable for the isolation ofexosomes. See Section 5.2. For example, the placenta-derived adherentcell exosomes described herein can be isolated by centrifugation (e.g.,density-dependent ultracentrifugation), polymer co-precipitation (e.g.,ExoQuick-TC exosome precipitation reagent by System Biosciences),filtration (e.g., high-performance liquid chromatography andgel-filtration chromatography), antibody capture, orfluorescence-activated cell sorting (FACS). In certain embodiments, theisolation and purification of the placenta-derived adherent cellexosomes provided herein results in a substantially pureplacenta-derived adherent cell exosome population, e.g., aplacenta-derived adherent cell exosome population that is at least 99%,98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% pure, e.g., as determinedby the presence of one or more markers associated with theplacenta-derived adherent cell exosomes provided herein (e.g., surfacemarkers and/or miRNAs). In a certain embodiment, the placenta-derivedadherent cell exosome population that is at least 99% pure. In a certainembodiment, the placenta-derived adherent cell exosome population thatis at least 98% pure. In a certain embodiment, the placenta-derivedadherent cell exosome population that is at least 97% pure. In a certainembodiment, the placenta-derived adherent cell exosome population thatis at least 96% pure. In a certain embodiment, the placenta-derivedadherent cell exosome population that is at least 95% pure. In a certainembodiment, the placenta-derived adherent cell exosome population thatis at least 94% pure. In a certain embodiment, the placenta-derivedadherent cell exosome population that is at least 93% pure. In a certainembodiment, the placenta-derived adherent cell exosome population thatis at least 92% pure. In a certain embodiment, the placenta-derivedadherent cell exosome population that is at least 91% pure. In a certainembodiment, the placenta-derived adherent cell exosome population thatis at least 90% pure.

Also provided herein are compositions comprising placenta-derivedadherent cell exosomes. Such compositions generally do not compriseplacental cells from which the placenta-derived adherent cell exosomeshave been derived. Moreover, such compositions generally do not comprisecell culture supernatant from the cell culture from which theplacenta-derived adherent cell exosomes have been derived.

In certain embodiments, purified placenta-derived adherent cell exosomesare formulated into pharmaceutical compositions suitable foradministration to a subject in need thereof. In certain embodiments,said subject is a human. The placenta-derived adherent cellexosome-containing pharmaceutical compositions provided herein can beformulated to be administered locally, systemically subcutaneously,parenterally, intravenously, intramuscularly, topically, orally,intradermally, transdermally, or intranasally to a subject in needthereof. In a certain embodiment, the placenta-derived adherent cellexosome-containing pharmaceutical compositions provided herein areformulated for local administration. In a certain embodiment, theplacenta-derived adherent cell exosome-containing pharmaceuticalcompositions provided herein are formulated for systemic subcutaneousadministration. In a certain embodiment, the placenta-derived adherentcell exosome-containing pharmaceutical compositions provided herein areformulated for parenteral administration. In a certain embodiment, theplacenta-derived adherent cell exosome-containing pharmaceuticalcompositions provided herein are formulated for intramuscularadministration. In a certain embodiment, the placenta-derived adherentcell exosome-containing pharmaceutical compositions provided herein areformulated for topical administration. In a certain embodiment, theplacenta-derived adherent cell exosome-containing pharmaceuticalcompositions provided herein are formulated for oral administration. Ina certain embodiment, the placenta-derived adherent cellexosome-containing pharmaceutical compositions provided herein areformulated for intradermal administration. In a certain embodiment, theplacenta-derived adherent cell exosome-containing pharmaceuticalcompositions provided herein are formulated for transdermaladministration. In a certain embodiment, the placenta-derived adherentcell exosome-containing pharmaceutical compositions provided herein areformulated for intranasal administration. In a specific embodiment, theplacenta-derived adherent cell exosome-containing pharmaceuticalcompositions provided herein are formulated for intravenousadministration.

In a specific embodiment, the placenta-derived adherent cells from whichthe exosomes provided herein are derived are CD34⁻, CD10⁺, CD105⁺ andCD200⁺. In another embodiment, the placenta-derived adherent cells fromwhich the exosomes provided herein are derived express one or more genesat a level at least two-fold higher than bone marrow-derived mesenchymalstem cells, wherein said one or more genes are one or more of: ACTG2,ADARB1, AMIGO2, ARTS-1, B4GALT6, BCHE, Cllorf9, CD200, COL4A1, COL4A2,CPA4, DMD, DSC3, DSG2, ELOVL2, F2RL1, FIJ10781, GATA6, GPR126, GPRC5B,HLA-G, ICAM1, IER3, IGFBP7, IL1A, IL6, IL18, KRT18, KRT8, LIPG, LRAP,MATN2, MEST, NFE2L3, NUAK1, PCDH7, PDLIM3, PJP2, RTN1, SERPINB9,ST3GAL6, ST6GALNAC5, SLC12A8, TCF21, TGFB2, VTN, and/or ZC3H12A. Inanother embodiment, the placenta-derived adherent cells from which theexosomes provided herein are derived express one or more genes at alevel at least two-fold higher than bone marrow-derived mesenchymal stemcells, wherein said one or more genes are one or more of LOVL2, ST3GAL6,ST6GALNAC5, and/or SLC12A8. In another embodiment, the placenta-derivedadherent cells from which the exosomes provided herein are derivedexpress one or more genes at a level at least two-fold higher than bonemarrow-derived mesenchymal stem cells, wherein said one or more genesare one or more of CPA4, TCF21, VTN, B4GALT6, FLJ10781, and/or NUAK1.

In certain embodiments, the placenta-derived adherent cell exosomesdescribed herein are isolated from placenta-derived adherent cellcultures that contain serum (e.g., fetal bovine serum). In certainembodiments, the placenta-derived adherent cell exosomes describedherein are isolated from placenta-derived adherent cell cultures thatlack serum.

In certain embodiments, the placenta-derived adherent cells from whichthe placenta-derived adherent cell exosomes described herein areisolated have been passaged at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12,14, 16, 18, or 20 times, or more, before said exosomes are isolated. Ina certain embodiment, the placenta-derived adherent cells have beenpassaged at least 1 time. In a certain embodiment, the placenta-derivedadherent cells have been passaged at least 2 times. In a certainembodiment, the placenta-derived adherent cells have been passaged atleast 3 times. In a certain embodiment, the placenta-derived adherentcells have been passaged at least 4 times. In a certain embodiment, theplacenta-derived adherent cells have been passaged at least 5 times. Ina certain embodiment, the placenta-derived adherent cells have beenpassaged at least 6 times. In a certain embodiment, the placenta-derivedadherent cells have been passaged at least 7 times. In a certainembodiment, the placenta-derived adherent cells have been passaged atleast 8 times. In a certain embodiment, the placenta-derived adherentcells have been passaged at least 9 times. In a certain embodiment, theplacenta-derived adherent cells have been passaged at least 10 times. Ina certain embodiment, the placenta-derived adherent cells have beenpassaged at least 12 times. In a certain embodiment, theplacenta-derived adherent cells have been passaged at least 14 times. Ina certain embodiment, the placenta-derived adherent cells have beenpassaged at least 16 times. In a certain embodiment, theplacenta-derived adherent cells have been passaged at least 18 times. Ina certain embodiment, the placenta-derived adherent cells have beenpassaged at least 20 times. In certain embodiments, the placenta-derivedadherent cells from which the placenta-derived adherent cell exosomesdescribed herein are isolated have been expanded for 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38 or40 population doublings, or more, before said exosomes are isolated. Ina certain embodiment, the placenta-derived adherent cells have beenexpanded for 1 population doubling. In a certain embodiment, theplacenta-derived adherent cells have been expanded for 2 populationdoublings. In a certain embodiment, the placenta-derived adherent cellshave been expanded for 3 population doublings. In a certain embodiment,the placenta-derived adherent cells have been expanded for 4 populationdoublings. In a certain embodiment, the placenta-derived adherent cellshave been expanded for 5 population doublings. In a certain embodiment,the placenta-derived adherent cells have been expanded for 6 populationdoublings. In a certain embodiment, the placenta-derived adherent cellshave been expanded for 7 population doublings. In a certain embodiment,the placenta-derived adherent cells have been expanded for 8 populationdoublings. In a certain embodiment, the placenta-derived adherent cellshave been expanded for 9 population doublings. In a certain embodiment,the placenta-derived adherent cells have been expanded for 10 populationdoublings. In a certain embodiment, the placenta-derived adherent cellshave been expanded for 12 population doublings. In a certain embodiment,the placenta-derived adherent cells have been expanded for 14 populationdoublings. In a certain embodiment, the placenta-derived adherent cellshave been expanded for 16 population doublings. In a certain embodiment,the placenta-derived adherent cells have been expanded for 18 populationdoublings. In a certain embodiment, the placenta-derived adherent cellshave been expanded for 20 population doublings. In a certain embodiment,the placenta-derived adherent cells have been expanded for 22 populationdoublings. In a certain embodiment, the placenta-derived adherent cellshave been expanded for 24 population doublings. In a certain embodiment,the placenta-derived adherent cells have been expanded for 26 populationdoublings. In a certain embodiment, the placenta-derived adherent cellshave been expanded for 28 population doublings. In a certain embodiment,the placenta-derived adherent cells have been expanded for 30 populationdoublings. In a certain embodiment, the placenta-derived adherent cellshave been expanded for 32 population doublings. In a certain embodiment,the placenta-derived adherent cells have been expanded for 34 populationdoublings. In a certain embodiment, the placenta-derived adherent cellshave been expanded for 36 population doublings. In a certain embodiment,the placenta-derived adherent cells have been expanded for 38 populationdoublings. In a certain embodiment, the placenta-derived adherent cellshave been expanded for 40 population doublings.

In certain embodiments, the placenta-derived adherent cells from whichthe placenta-derived adherent cell exosomes described herein areisolated are at least 80%, at least 90%, at least 95%, at least 99%, or100% fetal in origin. In a certain embodiment, the placenta-derivedadherent cells are at least 80% fetal in origin. In a certainembodiment, the placenta-derived adherent cells are at least 90% fetalin origin. In a certain embodiment, the placenta-derived adherent cellsare at least 95% fetal in origin. In a certain embodiment, theplacenta-derived adherent cells are at least 99% fetal in origin. In acertain embodiment, the placenta-derived adherent cells are at least100% fetal in origin.

In another aspect, provided herein are uses of the placenta-derivedadherent cell exosomes and/or pharmaceutical compositions comprisingplacenta-derived adherent cell exosomes described herein. See Section5.4.

In a specific embodiment, the placenta-derived adherent cell exosomesand/or pharmaceutical compositions comprising placenta-derived adherentcell exosomes described herein are used to treat and/or prevent diseasesand/or conditions in a subject in need thereof. In a specificembodiment, the placenta-derived adherent cell exosomes and/orpharmaceutical compositions comprising placenta-derived adherent cellexosomes described herein are used to promote angiogenesis and/orvascularization in a subject in need thereof. In another specificembodiment, the placenta-derived adherent cell exosomes and/orpharmaceutical compositions comprising placenta-derived adherent cellexosomes described herein are used to modulate immune activity (e.g.,increase an immune response or decrease an immune response) in a subjectin need thereof. In another specific embodiment, the placenta-derivedadherent cell exosomes and/or pharmaceutical compositions comprisingplacenta-derived adherent cell exosomes described herein are used torepair tissue damage, e.g., tissue damage caused by an acute or chronicinjury, in a subject in need thereof

In another specific embodiment, the derived adherent cell exosomesand/or pharmaceutical compositions comprising placenta-derived adherentcell exosomes described herein are for use in a method for treatingand/or preventing diseases and/or conditions in a subject in needthereof. In another embodiment, the pharmaceutical compositionscomprising placenta-derived adherent cell exosomes described herein arefor use in a method for treating diseases and/or conditions in a subjectin need thereof. In another embodiment, the pharmaceutical compositionscomprising placenta-derived adherent cell exosomes described herein arefor use in a method for preventing diseases and/or conditions in asubject in need thereof. In a specific embodiment, the pharmaceuticalcompositions comprising placenta-derived adherent cell exosomesdescribed herein are for use in a method for promoting angiogenesisand/or vascularization in a subject in need thereof. In another specificembodiment, the pharmaceutical compositions comprising placenta-derivedadherent cell exosomes described herein are for use in a method formodulating immune activity (e.g., increase an immune response ordecrease an immune response) in a subject in need thereof. In anotherspecific embodiment, the pharmaceutical compositions comprisingplacenta-derived adherent cell exosomes described herein are for use ina method for repairing tissue damage, e.g., tissue damage caused by anacute or chronic injury, in a subject in need thereof

In another specific embodiment, the placenta-derived adherent cellexosomes and/or pharmaceutical compositions comprising placenta-derivedadherent cell exosomes described herein are used as cytoprotectiveagents. In another aspect, the placenta-derived adherent cell exosomesand/or pharmaceutical compositions comprising placenta-derived adherentcell exosomes described herein are provided in the form of a kitsuitable for pharmaceutical use. See Section 5.5.

In another embodiment, provided herein are methods of loadingplacenta-derived adherent cell exosomes with exogenous agents, forexample, pharmaceutical agents. In one embodiment, such a methodcomprises incubating a placenta-derived adherent cell exosome and anexogenous agent, e.g., pharmaceutical agent, for example incubating atroom temperature, with or without saponin permeabilization, freeze/thawcycles, sonication, or extrusion. In certain embodiments, the exogenousagent, e.g., pharmaceutical agent, is loaded into the placenta-derivedadherent cell exosome by incubation at room temperature. In specificembodiments, incubation, e.g., incubation at room temperature, isperformed without saponin permeabilization of the exosome. In specificembodiments, incubation, e.g., incubation at room temperature, isperformed with saponin permeabilization of the placenta-derived adherentcell exosome. In certain embodiments, the exogenous agent, e.g.,pharmaceutical agent, is loaded into the exosome by freeze/thaw cycles,for example, 1, 2, 3, 4, 5, or more freeze/thaw cycles. In certainembodiments, the exogenous agent, e.g., pharmaceutical agent, is loadedinto the placenta-derived adherent cell exosome by sonication. Incertain embodiments, the exogenous agent, e.g., pharmaceutical agent, isloaded into the placenta-derived adherent cell exosome by extrusion. Incertain embodiments, the exogenous agent is loaded into theplacenta-derived adherent cell exosome by electroporation. In specificembodiments, methods of loading placenta-derived adherent cell exosomeswith exogenous agents, for example, pharmaceutical agents, comprise anycombination of the above.

In certain embodiments, the exogenous agent, e.g., pharmaceutical agent,loaded into the placenta-derived adherent cell exosomes described hereincomprises a polypeptide. In specific embodiments, the exogenous agent,e.g., pharmaceutical agent is a binding agent, for example, an antibody,such as, e.g., a human, humanized, or chimeric antibody, orantigen-binding fragment thereof. In particular embodiments, theantibody is a monospecific, bispecific or multispecific antibody, orantigen-binding fragment thereof. In yet other particular embodiments,the antibody or antigen binding fragment thereof is a single-chainantibody or a Fab fragment. In specific embodiments, the antibody orantigen-binding fragment thereof would not internalize into a targetcell, e.g., a cell other than the cell type from which the exosomes wereobtained, without the aid of the placenta-derived adherent cell exosome.

In certain embodiments, the exogenous agent, e.g., pharmaceutical agent,loaded into the placenta-derived adherent cell exosomes described hereincomprises one or more nucleic acids. In specific embodiments, the one ormore nucleic acids comprise a small interfering RNA (siRNA) or an miRNA.

In certain embodiments, the exogenous agent, e.g., pharmaceutical agent,loaded into the placenta-derived adherent cell exosomes described hereincomprises one or more gene-modifying components. In specificembodiments, the one or more gene-modifying components comprise aCRISPR-Cas system. In more specific embodiments, the CRISPR-Cas systemcomprises a guide RNA. In more specific embodiments, the CRISPR-Cassystem comprises an endonuclease. In more specific embodiments, theCRISPR-Cas system comprises a guide RNA and an endonuclease. In morespecific embodiments, the CRISPR-Cas system comprises Cas9. In morespecific embodiments, the CRISPR-Cas system comprises Cpf1. In specificembodiments, the one or more gene-modifying components comprise a zincfinger nuclease. In specific embodiments, the one or more gene-modifyingcomponents comprise a transcription activator-like effector nuclease(TALEN) system.

In another embodiment, provided herein are methods of delivering anexogenous agent, e.g., a pharmaceutical agent, to a target cell, whereinthe exogenous agent is loaded into a placenta-derived adherent cellexosome. In certain embodiments, the target cell is a cell other thanthe cell type from which the exosome was obtained. In certainembodiments, the exogenous agent, e.g., pharmaceutical agent, loadedinto the placenta-derived adherent cell exosomes described hereincomprises a polypeptide. In specific embodiments, the exogenous agent,e.g., pharmaceutical agent is a binding agent, for example, an antibody,such as, e.g., a human, humanized, or chimeric antibody, orantigen-binding fragment thereof. In particular embodiments, theantibody is a monospecific, bispecific or multispecific antibody, orantigen-binding fragment thereof. In yet other particular embodiments,the antibody or antigen binding fragment thereof is a single-chainantibody or a Fab fragment. In specific embodiments, the antibody orantigen-binding fragment thereof would not internalize into a targetcell, e.g., a cell other than the cell type from which the exosomes wereobtained, without the aid of the placenta-derived adherent cell exosome.In certain embodiments, the exogenous agent, e.g., pharmaceutical agent,loaded into the placenta-derived adherent cell exosomes described hereincomprises one or more nucleic acids. In specific embodiments, the one ormore nucleic acids comprise a siRNA or an miRNA. In certain embodiments,the exogenous agent, e.g., pharmaceutical agent, loaded into theplacenta-derived adherent cell exosomes described herein comprises oneor more gene-modifying components. In specific embodiments, the one ormore gene-modifying components comprise a CRISPR-Cas system. In morespecific embodiments, the CRISPR-Cas system comprises a guide RNA. Inmore specific embodiments, the CRISPR-Cas system comprises anendonuclease. In more specific embodiments, the CRISPR-Cas systemcomprises a guide RNA and an endonuclease. In more specific embodiments,the CRISPR-Cas system comprises Cas9. In more specific embodiments, theCRISPR-Cas system comprises Cpf1. In specific embodiments, the one ormore gene-modifying components comprise a zinc finger nuclease. Inspecific embodiments, the one or more gene-modifying components comprisea transcription activator-like effector nuclease (TALEN) system.

In another embodiment, provided herein are methods of administeringexosomes comprising exogenous agents, for example, pharmaceuticalagents, to a subject, e.g., a human. In certain embodiments, theexogenous agent, e.g., pharmaceutical agent, comprises a polypeptide. Inspecific embodiments, the exogenous agent, e.g., pharmaceutical agent,is a binding agent, for example, an antibody, such as, e.g., a human,humanized, or chimeric antibody, or antigen-binding fragment thereof. Inparticular embodiments, the antibody is a monospecific, bispecific ormultispecific antibody, or antigen-binding fragment thereof. In yetother particular embodiments, the antibody or antigen binding fragmentthereof is a single-chain antibody or a Fab fragment. In specificembodiments, the antibody or antigen-binding fragment thereof would notinternalize into a target cell without the aid of the exosome. Incertain embodiments, the exogenous agent, e.g., pharmaceutical agent,loaded into the placenta-derived adherent cell exosomes described hereincomprises one or more nucleic acids. In specific embodiments, the one ormore nucleic acids comprise a siRNA or an miRNA. In certain embodiments,the exogenous agent, e.g., pharmaceutical agent, loaded into theplacenta-derived adherent cell exosomes described herein comprises oneor more gene-modifying components. In specific embodiments, the one ormore gene-modifying components comprise a CRISPR-Cas system. In morespecific embodiments, the CRISPR-Cas system comprises a guide RNA. Inmore specific embodiments, the CRISPR-Cas system comprises anendonuclease. In more specific embodiments, the CRISPR-Cas systemcomprises a guide RNA and an endonuclease. In more specific embodiments,the CRISPR-Cas system comprises Cas9. In more specific embodiments, theCRISPR-Cas system comprises Cpf1. In specific embodiments, the one ormore gene-modifying components comprise a zinc finger nuclease. Inspecific embodiments, the one or more gene-modifying components comprisea transcription activator-like effector nuclease (TALEN) system.

In another embodiment, provided herein are compositions comprisingplacenta-derived adherent cell exosomes loaded with one or moreexogenous agents, for example, pharmaceutical agents. In certainembodiments, the exogenous agent, e.g., pharmaceutical agent,incorporated into the placenta-derived adherent cell exosomes describedherein comprises a polypeptide. In specific embodiments, the exogenousagent, e.g., pharmaceutical agent, is a binding agent, for example, anantibody, such as, e.g., a human, humanized, or chimeric antibody, orantigen-binding fragment thereof. In particular embodiments, theantibody is a monospecific, bispecific or multispecific antibody, orantigen-binding fragment thereof. In yet other particular embodiments,the antibody or antigen binding fragment thereof is a single-chainantibody or a Fab fragment. In specific embodiments, the antibody orantigen-binding fragment thereof would not internalize into a targetcell without the aid of the exosome. In certain embodiments, theexogenous agent, e.g., pharmaceutical agent, loaded into theplacenta-derived adherent cell exosomes described herein comprises oneor more nucleic acids. In specific embodiments, the one or more nucleicacids comprise a siRNA or an miRNA. In certain embodiments, theexogenous agent, e.g., pharmaceutical agent, loaded into theplacenta-derived adherent cell exosomes described herein comprises oneor more gene-modifying components. In specific embodiments, the one ormore gene-modifying components comprise a CRISPR-Cas system. In morespecific embodiments, the CRISPR-Cas system comprises a guide RNA. Inmore specific embodiments, the CRISPR-Cas system comprises anendonuclease. In more specific embodiments, the CRISPR-Cas systemcomprises a guide RNA and an endonuclease. In more specific embodiments,the CRISPR-Cas system comprises Cas9. In more specific embodiments, theCRISPR-Cas system comprises Cpf1. In specific embodiments, the one ormore gene-modifying components comprise a zinc finger nuclease. Inspecific embodiments, the one or more gene-modifying components comprisea transcription activator-like effector nuclease (TALEN) system.

4. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of an exosome antibody array demonstrating theexistence of placenta-derived adherent cell exosomes. Top panel:positive control from chorionic villi mesenchymal stem cells; bottompanel placenta-derived adherent cell exosomes.

FIG. 2A-2B: FIG. 2A is an electron micrograph of placenta-derivedadherent cell exosomes, with arrows indicating cup-shaped structuresthat are a common distinguishing feature of exosomes. FIG. 2B shows thesize distribution of placenta-derived adherent cell exosomes asdetermined by nanoparticle tracking assay.

FIGS. 3A-3D show the elution profiles of known molecules (FIG. 3A) ascompared to exosomes from either chorionic villi mesenchymal stem cells(FIG. 3B) or the non-chorionic placenta-derived adherent cells describedherein isolated under culture conditions containing or lacking serum(FIGS. 3C and 3D).

FIGS. 4A-4C: FIG. 4A shows levels of various microRNAs contained inexosomes isolated from chorionic villi mesenchymal stem cells or fromthe exosomes described herein. FIGS. 4B-4C show levels of variousangiogenic proteins contained on or within the exosomes describedherein.

FIG. 5 shows the ability for placenta-derived adherent cell exosomesdescribed herein to be taken up by certain cells of the blood.

FIG. 6 shows the effects of placenta-derived adherent cell exosomes andplacenta-derived adherent cells on the proliferation of human vascularendothelial cells.

FIGS. 7A-B show the levels of various cytokines and chemokines producedin vitro by LPS-stimulated human blood after incubation withplacenta-derived adherent cells or placenta-derived adherent cellexosomes.

FIGS. 8A-8G shows the effects of placenta-derived adherent cell exosomeson vascular endothelial cell growth and proliferation. FIGS. 8A and 8Bshow the ability of placenta-derived adherent cell exosomes to promotetube formation and branching of cultured vascular cells, when saidexosomes are collected from placenta-derived adherent cell cultureseither containing serum (FIG. 8A) or lacking serum (FIG. 8B). FIG. 8Cshows the relative expression of VEGF pathway genes after co-cultureswith placenta-derived adherent cell exosomes. Darker shades correspondwith higher expression, with the exception of the entries in white,which are positive controls (i.e., 18S, GAPDH, HPRT1, and GUSB). FIGS.8D-8G show the changes in gene expression for VEGF pathway genes overtime after co-culturing vascular endothelial cells with placenta-derivedadherent cells or placenta-derived adherent cell exosomes. CulturedHUVECs were incubated with placenta-derived adherent cell culturesupernatant (crosses), 50 μg of placenta-derived adherent cell exosomes(triangles), or culture medium (diamonds) as a control.

FIGS. 9A-9B: FIG. 9A shows the relative levels of TNF-α and MCP-1secreted by monocyte-derived macrophages after co-culture withplacenta-derived adherent cells or placenta-derived adherent cellexosomes. FIG. 9B shows the level of secretion of TNF-α and IL-8 (leftand middle, respectively) secreted by macrophages after co-culture withplacenta-derived adherent cells or placenta-derived adherent cellexosomes. Also shown (FIG. 9B, right) is the effect of intact or lysedplacenta-derived adherent cell exosomes on IL-8 secretion frommonocyte-derived macrophages.

5. DETAILED DESCRIPTION

5.1. Placenta-Derived Adherent Cell Exosomes

The placenta-derived adherent cell exosomes described herein can beselected and identified by their morphology and/or molecular markers, asdescribed below. The placenta-derived adherent cell exosomes describedherein are distinct from exosomes known in the art e.g., chorionic villimesenchymal stem cell-derived exosomes, e.g., those described in Salomonet al., 2013, PLOS ONE, 8:7, e68451. Accordingly, the term“placenta-derived adherent cell exosome,” as used herein, is not meantto include exosomes obtained or derived from chorionic villi mesenchymalstem cells.

In certain embodiments, populations of placenta-derived adherent cellexosomes described herein do not comprise cells, e.g., nucleated cells,for example placental cells, e.g., placenta-derived adherent cells.

5.1.1. Placenta-Derived Adherent Cell Exosome Markers

The placenta-derived adherent cell exosomes described herein containmarkers that can be used to identify and/or isolate said exosomes. Thesemarkers may, for example, be proteins, nucleic acids, saccharidemolecules, glycosylated proteins, lipid molecules, and may exist inmonomeric, oligomeric and/or multimeric form. In certain embodiments,the markers are produced by the placenta-derived adherent cell fromwhich the exosomes are derived. In certain embodiments, the marker isprovided by the placenta-derived adherent cell from which the exosomesare derived, but the marker is not expressed at a higher level by saidcell. In a specific embodiment, the markers of placenta-derived adherentcell exosomes described herein are higher in the exosomes as compared tothe cell of origin when compared to a control marker molecule. Inanother specific embodiment, the markers of placenta-derived adherentcell exosomes described herein are enriched in said exosomes as comparedto exosomes obtained from another cell type (e.g., the chorionic villimesenchymal stem cells described in Salomon et al., 2013, PLOS ONE, 8:7,e68451 and pre-adipocyte mesenchymal stem cells), wherein the exosomesare obtained through identical methods and wherein the cells from whichthe exosomes are derived are maintained under identical conditions.

The three-dimensional structure of exosomes allows for the retention ofmarkers on the surface of the exosome and/or contained within theexosome. Similarly, marker molecules may exist partially within theexosome, partially on the outer surface of the exosome and/or across thephospholipid bilayer of the exosome. In a specific embodiment, themarkers associated with the placenta-derived adherent cell exosomesdescribed herein are proteins. In certain embodiments, the markers aretransmembrane proteins that are anchored within the exosome phospholipidbilayer, or are anchored across the exosome phospholipid bilayer suchthat portions of the protein molecule are within the exosome whileportions of the same molecule are exposed to the outer surface of theexosome. In certain embodiments, the markers are contained entirelywithin the exosome. In another specific embodiment, the markersassociated with the placenta-derived adherent cell exosomes describedherein are nucleic acids. In certain embodiments, said nucleic acids arenon-coding RNA molecules, e.g., micro-RNAs (miRNAs).

5.1.1.1. Surface Markers

The placenta-derived adherent cell exosomes described herein comprisesurface markers that allow for their identification and that can be usedto isolate/obtain substantially pure populations of placenta-derivedadherent cell exosomes free from their placenta-derived adherent cellsof origin and other cellular and non-cellular material. Methods of fordetermining exosome surface marker composition are known in the art. Forexample, exosomal surface markers can be detected byfluorescence-activated cell sorting (FACS) or Western blotting.

The placenta-derived adherent cell exosomes described herein containdistinct surface markers that allow them to be distinguished from otherexosomes known in the art. In a specific embodiment, theplacenta-derived adherent cell exosomes provided herein comprise one,two, three, four, five, or more of the following markers: CD9, CD10,CD13, CD29, CD44, CD49b, CD49c, CD55, CD59, CD63, CD73, CD81, CD82,CD90, CD98, CD105, CD141, CD142, CD151, CD164, CD295, CD200 (asdeterminable by, e.g., FACS).

In one specific embodiment, the placenta-derived adherent cell exosomesdescribed herein are CD55⁺ and CD10⁺ as determined, e.g., by flowcytometry, for example, by FACS.

In specific embodiments, the placenta-derived adherent cell exosomesdescribed herein are CD10⁺ and CD55⁺ (as determined by, e.g., FACS) andadditionally contain CD82, CD142, CD49c, and CD90 at a level higher thanchorionic villi mesenchymal stem cell exosomes or pre-adipocytemesenchymal stem cell exosomes (e.g., the chorionic villi mesenchymalstem cells described in Salomon et al., 2013, PLOS ONE, 8:7, e68451).

In a specific embodiment, the placenta-derived adherent cell exosomesdescribed herein are CD9⁺. In another specific embodiment, theplacenta-derived adherent cell exosomes described herein are CD10⁺. Inanother specific embodiment, the placenta-derived adherent cell exosomesdescribed herein are CD13⁺. In another specific embodiment, theplacenta-derived adherent cell exosomes described herein are CD29⁺. Inanother specific embodiment, the placenta-derived adherent cell exosomesdescribed herein are CD44⁺. In another specific embodiment, theplacenta-derived adherent cell exosomes described herein are CD49b⁺. Inanother specific embodiment, the placenta-derived adherent cell exosomesdescribed herein are CD49c⁺. In another specific embodiment, theplacenta-derived adherent cell exosomes described herein are CD55⁺. Inanother specific embodiment, the placenta-derived adherent cell exosomesdescribed herein are CD59⁺. In another specific embodiment, theplacenta-derived adherent cell exosomes described herein are CD63⁺. Inanother specific embodiment, the placenta-derived adherent cell exosomesdescribed herein are CD73⁺. In another specific embodiment, theplacenta-derived adherent cell exosomes described herein are CD81⁺. Inanother specific embodiment, the placenta-derived adherent cell exosomesdescribed herein are CD82⁺. In another specific embodiment, theplacenta-derived adherent cell exosomes described herein are CD90⁺. Inanother specific embodiment, the placenta-derived adherent cell exosomesdescribed herein are CD98⁺. In another specific embodiment, theplacenta-derived adherent cell exosomes described herein are CD105⁺. Inanother specific embodiment, the placenta-derived adherent cell exosomesdescribed herein are CD141⁺. In another specific embodiment, theplacenta-derived adherent cell exosomes described herein are CD142⁺. Inanother specific embodiment, the placenta-derived adherent cell exosomesdescribed herein are CD151⁺. In another specific embodiment, theplacenta-derived adherent cell exosomes described herein are CD164⁺. Inanother specific embodiment, the placenta-derived adherent cell exosomesdescribed herein are CD295⁺. In another specific embodiment, theplacenta-derived adherent cell exosomes described herein are CD200⁺.Determination of the presence of such markers can be made, e.g., by flowcytometry, for example, by FACS. In another embodiment, described hereinare populations of placenta-derived adherent cell exosomes containingany combination of the above-referenced surface markers.

In a specific embodiment, the placenta-derived adherent cell exosomesdescribed herein are CD9⁺ and additionally comprise one or more of themarkers selected from the group of CD10, CD13, CD29, CD44, CD49b, CD49c,CD55, CD59, CD63, CD73, CD81, CD82, CD90, CD98, CD105, CD141, CD142,CD151, CD164, CD295, and CD200.

In another specific embodiment, the placenta-derived adherent cellexosomes described herein are CD10⁺ and additionally comprise one ormore of the markers selected from the group of CD9, CD13, CD29, CD44,CD49b, CD49c, CD55, CD59, CD63, CD73, CD81, CD82, CD90, CD98, CD105,CD141, CD142, CD151, CD164, CD295, and CD200.

In another specific embodiment, the placenta-derived adherent cellexosomes described herein are CD13⁺ and additionally comprise one ormore of the markers selected from the group of CD9, CD10, CD29, CD44,CD49b, CD49c, CD55, CD59, CD63, CD73, CD81, CD82, CD90, CD98, CD105,CD141, CD142, CD151, CD164, CD295, and CD200.

In another specific embodiment, the placenta-derived adherent cellexosomes described herein are CD29⁺ and additionally comprise one ormore of the markers selected from the group of CD9, CD10, CD13, CD44,CD49b, CD49c, CD55, CD59, CD63, CD73, CD81, CD82, CD90, CD98, CD105,CD141, CD142, CD151, CD164, CD295, and CD200.

In another specific embodiment, the placenta-derived adherent cellexosomes described herein are CD44⁺ and additionally comprise one ormore of the markers selected from the group of CD9, CD10, CD13, CD29,CD49b, CD49c, CD55, CD59, CD63, CD73, CD81, CD82, CD90, CD98, CD105,CD141, CD142, CD151, CD164, CD295, and CD200.

In another specific embodiment, the placenta-derived adherent cellexosomes described herein are CD49b⁺ and additionally comprise one ormore of the markers selected from the group of CD9, CD10, CD13, CD29,CD44, CD49c, CD55, CD59, CD63, CD73, CD81, CD82, CD90, CD98, CD105,CD141, CD142, CD151, CD164, CD295, and CD200.

In another specific embodiment, the placenta-derived adherent cellexosomes described herein are CD49c′ and additionally comprise one ormore of the markers selected from the group of CD9, CD10, CD13, CD29,CD44, CD49b, CD55, CD59, CD63, CD73, CD81, CD82, CD90, CD98, CD105,CD141, CD142, CD151, CD164, CD295, and CD200.

In another specific embodiment, the placenta-derived adherent cellexosomes described herein are CD55⁺ and additionally comprise one ormore of the markers selected from the group of CD9, CD10, CD13, CD29,CD44, CD49b, CD49c, CD59, CD63, CD73, CD81, CD82, CD90, CD98, CD105,CD141, CD142, CD151, CD164, CD295, and CD200.

In another specific embodiment, the placenta-derived adherent cellexosomes described herein are CD59⁺ and additionally comprise one ormore of the markers selected from the group of CD9, CD10, CD13, CD29,CD44, CD49b, CD49c, CD55, CD63, CD73, CD81, CD82, CD90, CD98, CD105,CD141, CD142, CD151, CD164, CD295, and CD200.

In another specific embodiment, the placenta-derived adherent cellexosomes described herein are CD63⁺ and additionally comprise one ormore of the markers selected from the group of CD9, CD10, CD13, CD29,CD44, CD49b, CD49c, CD55, CD59, CD73, CD81, CD82, CD90, CD98, CD105,CD141, CD142, CD151, CD164, CD295, and CD200.

In another specific embodiment, the placenta-derived adherent cellexosomes described herein are CD73⁺ and additionally comprise one ormore of the markers selected from the group of CD9, CD10, CD13, CD29,CD44, CD49b, CD49c, CD55, CD59, CD63, CD81, CD82, CD90, CD98, CD105,CD141, CD142, CD151, CD164, CD295, and CD200.

In another specific embodiment, the placenta-derived adherent cellexosomes described herein are CD81⁺ and additionally comprise one ormore of the markers selected from the group of CD9, CD10, CD13, CD29,CD44, CD49b, CD49c, CD55, CD59, CD63, CD73, CD82, CD90, CD98, CD105,CD141, CD142, CD151, CD164, CD295, and CD200.

In another specific embodiment, the placenta-derived adherent cellexosomes described herein are CD82⁺ and additionally comprise one ormore of the markers selected from the group of CD9, CD10, CD13, CD29,CD44, CD49b, CD49c, CD55, CD59, CD63, CD73, CD81, CD90, CD98, CD105,CD141, CD142, CD151, CD164, CD295, and CD200.

In another specific embodiment, the placenta-derived adherent cellexosomes described herein are CD90⁺ and additionally comprise one ormore of the markers selected from the group of CD9, CD10, CD13, CD29,CD44, CD49b, CD49c, CD55, CD59, CD63, CD73, CD81, CD82, CD98, CD105,CD141, CD142, CD151, CD164, CD295, and CD200.

In another specific embodiment, the placenta-derived adherent cellexosomes described herein are CD98⁺ and additionally comprise one ormore of the markers selected from the group of CD9, CD10, CD13, CD29,CD44, CD49b, CD49c, CD55, CD59, CD63, CD73, CD81, CD82, CD90, CD105,CD141, CD142, CD151, CD164, CD295, and CD200.

In another specific embodiment, the placenta-derived adherent cellexosomes described herein are CD105⁺ and additionally comprise one ormore of the markers selected from the group of CD9, CD10, CD13, CD29,CD44, CD49b, CD49c, CD55, CD59, CD63, CD73, CD81, CD82, CD90, CD98,CD141, CD142, CD151, CD164, CD295, and CD200.

In another specific embodiment, the placenta-derived adherent cellexosomes described herein are CD141⁺ and additionally comprise one ormore of the markers selected from the group of CD9, CD10, CD13, CD29,CD44, CD49b, CD49c, CD55, CD59, CD63, CD73, CD81, CD82, CD90, CD98,CD105, CD142, CD151, CD164, CD295, and CD200.

In another specific embodiment, the placenta-derived adherent cellexosomes described herein are CD142⁺ and additionally comprise one ormore of the markers selected from the group of CD9, CD10, CD13, CD29,CD44, CD49b, CD49c, CD55, CD59, CD63, CD73, CD81, CD82, CD90, CD98,CD105, CD141, CD151, CD164, CD295, and CD200.

In another specific embodiment, the placenta-derived adherent cellexosomes described herein are CD151⁺ and additionally comprise one ormore of the markers selected from the group of CD9, CD10, CD13, CD29,CD44, CD49b, CD49c, CD55, CD59, CD63, CD73, CD81, CD82, CD90, CD98,CD105, CD141, CD142, CD164, CD295, and CD200.

In another specific embodiment, the placenta-derived adherent cellexosomes described herein are CD164⁺ and additionally comprise one ormore of the markers selected from the group of CD9, CD10, CD13, CD29,CD44, CD49b, CD49c, CD55, CD59, CD63, CD73, CD81, CD82, CD90, CD98,CD105, CD141, CD142, CD151, CD295, and CD200.

In another specific embodiment, the placenta-derived adherent cellexosomes described herein are CD295⁺ and additionally comprise one ormore of the markers selected from the group of CD9, CD10, CD13, CD29,CD44, CD49b, CD49c, CD55, CD59, CD63, CD73, CD81, CD82, CD90, CD98,CD105, CD141, CD142, CD151, and CD200.

In another specific embodiment, the placenta-derived adherent cellexosomes described herein are CD200⁺ and additionally comprise one ormore of the markers selected from the group of CD9, CD10, CD13, CD29,CD44, CD49b, CD49c, CD55, CD59, CD63, CD73, CD81, CD82, CD90, CD98,CD105, CD141, CD142, CD164, and CD295.

In another specific embodiment, the placenta-derived adherent cellexosomes described herein are CD49b⁺, CD49c⁺, CD98⁺, CD29⁺, CD142⁺,CD10⁺, CD47⁺, CD55⁺, CD90⁺, CD147⁺, CD151⁺, CD166⁺, CD82⁺ and CD200⁺.

In another embodiment, described herein are populations ofplacenta-derived adherent cell exosomes containing any combination ofthe above-referenced surface markers.

The placenta-derived adherent cell exosomes described herein generallydo not comprise the surface markers CD3, CD11b, CD14, CD19, CD33, CD192,HLA-A, HLA-B, HLA-C, HLA-DR, CD11c or CD34. Thus, in one embodiment, theplacenta-derived adherent cell exosomes described herein are CD3⁻. Inanother embodiment, the placenta-derived adherent cell exosomesdescribed herein are CD11b⁻. In another embodiment, the placenta-derivedadherent cell exosomes described herein are CD14⁻. In anotherembodiment, the placenta-derived adherent cell exosomes described hereinare CD19⁻. In another embodiment, the placenta-derived adherent cellexosomes described herein are CD33⁻. In another embodiment, theplacenta-derived adherent cell exosomes described herein are CD192⁻. Inanother embodiment, the placenta-derived adherent cell exosomesdescribed herein are HLA-A⁻. In another embodiment, the placenta-derivedadherent cell exosomes described herein are HLA-B⁻. In anotherembodiment, the placenta-derived adherent cell exosomes described hereinare HLA-C⁻. In another embodiment, the placenta-derived adherent cellexosomes described herein are HLA-DR⁻. In another embodiment, theplacenta-derived adherent cell exosomes described herein are CD11c⁻. Inanother embodiment, the placenta-derived adherent cell exosomesdescribed herein are CD34⁻.

In a specific embodiment, the placenta-derived adherent cell exosomesdescribed herein are CD3⁻ and do not comprise one or more of the markersselected from the group of CD11b, CD14, CD19, CD33, CD192, HLA-A, HLA-B,HLA-C, HLA-DR, CD11c and CD34.

In another specific embodiment, the placenta-derived adherent cellexosomes described herein are CD11b⁻ and do not comprise one or more ofthe markers selected from the group of CD3, CD14, CD19, CD33, CD192,HLA-A, HLA-B, HLA-C, HLA-DR, CD11c and CD34.

In another specific embodiment, the placenta-derived adherent cellexosomes described herein are CD14⁻ and do not comprise one or more ofthe markers selected from the group of CD3, CD11b, CD19, CD33, CD192,HLA-A, HLA-B, HLA-C, HLA-DR, CD11c and CD34.

In another specific embodiment, the placenta-derived adherent cellexosomes described herein are CD19⁻ and do not comprise one or more ofthe markers selected from the group of CD3, CD11b, CD14, CD33, CD192,HLA-A, HLA-B, HLA-C, HLA-DR, CD11c and CD34.

In another specific embodiment, the placenta-derived adherent cellexosomes described herein are CD33⁻ and do not comprise one or more ofthe markers selected from the group of CD3, CD11b, CD14, CD19, CD192,HLA-A, HLA-B, HLA-C, HLA-DR, CD11c and CD34.

In another specific embodiment, the placenta-derived adherent cellexosomes described herein are CD192⁻ and do not comprise one or more ofthe markers selected from the group of CD3, CD11b, CD14, CD19, CD33,HLA-A, HLA-B, HLA-C, HLA-DR, CD11c and CD34.

In another specific embodiment, the placenta-derived adherent cellexosomes described herein are HLA-A⁻ and do not comprise one or more ofthe markers selected from the group of CD3, CD11b, CD14, CD19, CD33,CD192, HLA-B, HLA-C, HLA-DR, CD11c and CD34.

In another specific embodiment, the placenta-derived adherent cellexosomes described herein are HLA-B⁻ and do not comprise one or more ofthe markers selected from the group of CD3, CD11b, CD14, CD19, CD33,CD192, HLA-A, HLA-C, HLA-DR, CD11c and CD34.

In another specific embodiment, the placenta-derived adherent cellexosomes described herein are HLA-C⁻ and do not comprise one or more ofthe markers selected from the group of CD3, CD11b, CD14, CD19, CD33,CD192, HLA-A, HLA-B, HLA-DR, CD11c and CD34.

In another specific embodiment, the placenta-derived adherent cellexosomes described herein are HLA-DR⁻ and do not comprise one or more ofthe markers selected from the group of CD3, CD11b, CD14, CD19, CD33,CD192, HLA-A, HLA-B, HLA-C, CD11c and CD34.

In another specific embodiment, the placenta-derived adherent cellexosomes described herein are CD11c and do not comprise one or more ofthe markers selected from the group of CD3, CD11b, CD14, CD19, CD33,CD192, HLA-A, HLA-B, HLA-C, HLA-DR and CD34.

In another specific embodiment, the placenta-derived adherent cellexosomes described herein are CD34⁻ and do not comprise one or more ofthe markers selected from the group of CD3, CD11b, CD14, CD19, CD33,CD192, HLA-A, HLA-B, HLA-C, HLA-DR and CD11c.

In another specific embodiment, the placenta-derived adherent cellexosomes described herein are CD3⁻, CD11b⁻, CD14⁻, CD19⁻, CD33⁻, CD192⁻,HLA-A⁻, HLA-B⁻, HLA-C, HLA-DR⁻, CD11c⁻ and CD34⁻.

In another specific embodiment, the placenta-derived adherent cellexosomes described herein are CD9+ and additionally comprise one or moreof the markers selected from the group of CD10, CD13, CD29, CD44, CD49b,CD49c, CD55, CD59, CD63, CD73, CD81, CD82, CD90, CD98, CD105, CD141,CD142, CD151, CD164, CD295, and CD200, while further lacking one or moreof the markers selected from the group of CD3, CD11b, CD14, CD19, CD33,CD192, HLA-A, HLA-B, HLA-C, HLA-DR, CD11c and CD34.

In another specific embodiment, the placenta-derived adherent cellexosomes described herein are CD10+ and additionally comprise one ormore of the markers selected from the group of CD9, CD13, CD29, CD44,CD49b, CD49c, CD55, CD59, CD63, CD73, CD81, CD82, CD90, CD98, CD105,CD141, CD142, CD151, CD164, CD295, and CD200, while further lacking oneor more of the markers selected from the group of CD3, CD11b, CD14,CD19, CD33, CD192, HLA-A, HLA-B, HLA-C, HLA-DR, CD11c and CD34.

In another specific embodiment, the placenta-derived adherent cellexosomes described herein are CD13+ and additionally comprise one ormore of the markers selected from the group of CD9, CD10, CD29, CD44,CD49b, CD49c, CD55, CD59, CD63, CD73, CD81, CD82, CD90, CD98, CD105,CD141, CD142, CD151, CD164, CD295, and CD200, while further lacking oneor more of the markers selected from the group of CD3, CD11b, CD14,CD19, CD33, CD192, HLA-A, HLA-B, HLA-C, HLA-DR, CD11c and CD34.

In another specific embodiment, the placenta-derived adherent cellexosomes described herein are CD29+ and additionally comprise one ormore of the markers selected from the group of CD9, CD10, CD13, CD44,CD49b, CD49c, CD55, CD59, CD63, CD73, CD81, CD82, CD90, CD98, CD105,CD141, CD142, CD151, CD164, CD295, and CD200, while further lacking oneor more of the markers selected from the group of CD3, CD11b, CD14,CD19, CD33, CD192, HLA-A, HLA-B, HLA-C, HLA-DR, CD11c and CD34.

In another specific embodiment, the placenta-derived adherent cellexosomes described herein are CD44+ and additionally comprise one ormore of the markers selected from the group of CD9, CD10, CD13, CD29,CD49b, CD49c, CD55, CD59, CD63, CD73, CD81, CD82, CD90, CD98, CD105,CD141, CD142, CD151, CD164, CD295, and CD200, while further lacking oneor more of the markers selected from the group of CD3, CD11b, CD14,CD19, CD33, CD192, HLA-A, HLA-B, HLA-C, HLA-DR, CD11c and CD34.

In another specific embodiment, the placenta-derived adherent cellexosomes described herein are CD49b+ and additionally comprise one ormore of the markers selected from the group of CD9, CD10, CD13, CD29,CD44, CD49c, CD55, CD59, CD63, CD73, CD81, CD82, CD90, CD98, CD105,CD141, CD142, CD151, CD164, CD295, and CD200, while further lacking oneor more of the markers selected from the group of CD3, CD11b, CD14,CD19, CD33, CD192, HLA-A, HLA-B, HLA-C, HLA-DR, CD11c and CD34.

In another specific embodiment, the placenta-derived adherent cellexosomes described herein are CD49c+ and additionally comprise one ormore of the markers selected from the group of CD9, CD10, CD13, CD29,CD44, CD49b, CD55, CD59, CD63, CD73, CD81, CD82, CD90, CD98, CD105,CD141, CD142, CD151, CD164, CD295, and CD200, while further lacking oneor more of the markers selected from the group of CD3, CD11b, CD14,CD19, CD33, CD192, HLA-A, HLA-B, HLA-C, HLA-DR, CD11c and CD34.

In another specific embodiment, the placenta-derived adherent cellexosomes described herein are CD55+ and additionally comprise one ormore of the markers selected from the group of CD9, CD10, CD13, CD29,CD44, CD49b, CD49c, CD59, CD63, CD73, CD81, CD82, CD90, CD98, CD105,CD141, CD142, CD151, CD164, CD295, and CD200, while further lacking oneor more of the markers selected from the group of CD3, CD11b, CD14,CD19, CD33, CD192, HLA-A, HLA-B, HLA-C, HLA-DR, CD11c and CD34.

In another specific embodiment, the placenta-derived adherent cellexosomes described herein are CD59+ and additionally comprise one ormore of the markers selected from the group of CD9, CD10, CD13, CD29,CD44, CD49b, CD49c, CD55, CD63, CD73, CD81, CD82, CD90, CD98, CD105,CD141, CD142, CD151, CD164, CD295, and CD200, while further lacking oneor more of the markers selected from the group of CD3, CD11b, CD14,CD19, CD33, CD192, HLA-A, HLA-B, HLA-C, HLA-DR, CD11c and CD34.

In another specific embodiment, the placenta-derived adherent cellexosomes described herein are CD63+ and additionally comprise one ormore of the markers selected from the group of CD9, CD10, CD13, CD29,CD44, CD49b, CD49c, CD55, CD59, CD73, CD81, CD82, CD90, CD98, CD105,CD141, CD142, CD151, CD164, CD295, and CD200, while further lacking oneor more of the markers selected from the group of CD3, CD11b, CD14,CD19, CD33, CD192, HLA-A, HLA-B, HLA-C, HLA-DR, CD11c and CD34.

In another specific embodiment, the placenta-derived adherent cellexosomes described herein are CD73+ and additionally comprise one ormore of the markers selected from the group of CD9, CD10, CD13, CD29,CD44, CD49b, CD49c, CD55, CD59, CD63, CD81, CD82, CD90, CD98, CD105,CD141, CD142, CD151, CD164, CD295, and CD200, while further lacking oneor more of the markers selected from the group of CD3, CD11b, CD14,CD19, CD33, CD192, HLA-A, HLA-B, HLA-C, HLA-DR, CD11c and CD34.

In another specific embodiment, the placenta-derived adherent cellexosomes described herein are CD81+ and additionally comprise one ormore of the markers selected from the group of CD9, CD10, CD13, CD29,CD44, CD49b, CD49c, CD55, CD59, CD63, CD73, CD82, CD90, CD98, CD105,CD141, CD142, CD151, CD164, CD295, and CD200, while further lacking oneor more of the markers selected from the group of CD3, CD11b, CD14,CD19, CD33, CD192, HLA-A, HLA-B, HLA-C, HLA-DR, CD11c and CD34.

In another specific embodiment, the placenta-derived adherent cellexosomes described herein are CD82+ and additionally comprise one ormore of the markers selected from the group of CD9, CD10, CD13, CD29,CD44, CD49b, CD49c, CD55, CD59, CD63, CD73, CD81, CD90, CD98, CD105,CD141, CD142, CD151, CD164, CD295, and CD200, while further lacking oneor more of the markers selected from the group of CD3, CD11b, CD14,CD19, CD33, CD192, HLA-A, HLA-B, HLA-C, HLA-DR, CD11c and CD34.

In another specific embodiment, the placenta-derived adherent cellexosomes described herein are CD90+ and additionally comprise one ormore of the markers selected from the group of CD9, CD10, CD13, CD29,CD44, CD49b, CD49c, CD55, CD59, CD63, CD73, CD81, CD82, CD98, CD105,CD141, CD142, CD151, CD164, CD295, and CD200, while further lacking oneor more of the markers selected from the group of CD3, CD11b, CD14,CD19, CD33, CD192, HLA-A, HLA-B, HLA-C, HLA-DR, CD11c and CD34.

In another specific embodiment, the placenta-derived adherent cellexosomes described herein are CD98+ and additionally comprise one ormore of the markers selected from the group of CD9, CD10, CD13, CD29,CD44, CD49b, CD49c, CD55, CD59, CD63, CD73, CD81, CD82, CD90, CD105,CD141, CD142, CD151, CD164, CD295, and CD200, while further lacking oneor more of the markers selected from the group of CD3, CD11b, CD14,CD19, CD33, CD192, HLA-A, HLA-B, HLA-C, HLA-DR, CD11c and CD34.

In another specific embodiment, the placenta-derived adherent cellexosomes described herein are CD105+ and additionally comprise one ormore of the markers selected from the group of CD9, CD10, CD13, CD29,CD44, CD49b, CD49c, CD55, CD59, CD63, CD73, CD81, CD82, CD90, CD98,CD141, CD142, CD151, CD164, CD295, and CD200, while further lacking oneor more of the markers selected from the group of CD3, CD11b, CD14,CD19, CD33, CD192, HLA-A, HLA-B, HLA-C, HLA-DR, CD11c and CD34.

In another specific embodiment, the placenta-derived adherent cellexosomes described herein are CD141+ and additionally comprise one ormore of the markers selected from the group of CD9, CD10, CD13, CD29,CD44, CD49b, CD49c, CD55, CD59, CD63, CD73, CD81, CD82, CD90, CD98,CD105, CD142, CD151, CD164, CD295, and CD200, while further lacking oneor more of the markers selected from the group of CD3, CD11b, CD14,CD19, CD33, CD192, HLA-A, HLA-B, HLA-C, HLA-DR, CD11c and CD34.

In another specific embodiment, the placenta-derived adherent cellexosomes described herein are CD142+ and additionally comprise one ormore of the markers selected from the group of CD9, CD10, CD13, CD29,CD44, CD49b, CD49c, CD55, CD59, CD63, CD73, CD81, CD82, CD90, CD98,CD105, CD141, CD151, CD164, CD295, and CD200, while further lacking oneor more of the markers selected from the group of CD3, CD11b, CD14,CD19, CD33, CD192, HLA-A, HLA-B, HLA-C, HLA-DR, CD11c and CD34.

In another specific embodiment, the placenta-derived adherent cellexosomes described herein are CD151+ and additionally comprise one ormore of the markers selected from the group of CD9, CD10, CD13, CD29,CD44, CD49b, CD49c, CD55, CD59, CD63, CD73, CD81, CD82, CD90, CD98,CD105, CD141, CD142, CD164, CD295, and CD200, while further lacking oneor more of the markers selected from the group of CD3, CD11b, CD14,CD19, CD33, CD192, HLA-A, HLA-B, HLA-C, HLA-DR, CD11c and CD34.

In another specific embodiment, the placenta-derived adherent cellexosomes described herein are CD164+ and additionally comprise one ormore of the markers selected from the group of CD9, CD10, CD13, CD29,CD44, CD49b, CD49c, CD55, CD59, CD63, CD73, CD81, CD82, CD90, CD98,CD105, CD141, CD142, CD151, CD295, and CD200, while further lacking oneor more of the markers selected from the group of CD3, CD11b, CD14,CD19, CD33, CD192, HLA-A, HLA-B, HLA-C, HLA-DR, CD11c and CD34.

In another specific embodiment, the placenta-derived adherent cellexosomes described herein are CD295+ and additionally comprise one ormore of the markers selected from the group of CD9, CD10, CD13, CD29,CD44, CD49b, CD49c, CD55, CD59, CD63, CD73, CD81, CD82, CD90, CD98,CD105, CD141, CD142, CD151, and CD200, while further lacking one or moreof the markers selected from the group of CD3, CD11b, CD14, CD19, CD33,CD192, HLA-A, HLA-B, HLA-C, HLA-DR, CD11c and CD34.

In another specific embodiment, the placenta-derived adherent cellexosomes described herein are CD200+ and additionally comprise one ormore of the markers selected from the group of CD9, CD10, CD13, CD29,CD44, CD49b, CD49c, CD55, CD59, CD63, CD73, CD81, CD82, CD90, CD98,CD105, CD141, CD142, CD164, and CD295, while further lacking one or moreof the markers selected from the group of CD3, CD11b, CD14, CD19, CD33,CD192, HLA-A, HLA-B, HLA-C, HLA-DR, CD11c and CD34.

In another embodiment, described herein are populations ofplacenta-derived adherent cell exosomes containing any combination ofthe above-referenced surface markers.

In another specific embodiment, the placenta-derived adherent cellexosomes described herein are CD9⁺, CD10⁺, CD13⁺, CD29⁺, CD44⁺, CD49b⁺,CD49c′, CD55⁺, CD59⁺, CD63⁺, CD73⁺, CD81⁺, CD82⁺, CD90⁺, CD98⁺, CD105⁺,CD141⁺, CD142⁺, CD151⁺, CD164⁺, CD295⁺, and CD200⁺ while further lackingone or more of the markers selected from the group of CD3, CD11b, CD14,CD19, CD33, CD192, HLA-A, HLA-B, HLA-C, HLA-DR, CD11c and CD34.

In another specific embodiment, the placenta-derived adherent cellexosomes described herein are CD3⁻, CD11b⁻, CD14⁻, CD19⁻, CD33⁻, CD192⁻,HLA-A⁻, HLA-B⁻, HLA-C⁻, HLA-DR⁻, CD11c⁻ and CD34⁻ while additionallycomprising one or more of the markers selected from the group of CD9,CD10, CD13, CD29, CD44, CD49b, CD49c, CD55, CD59, CD63, CD73, CD81,CD82, CD90, CD98, CD105, CD141, CD142, CD151, CD164, CD295, and CD200.

In another specific embodiment, the placenta-derived adherent cellexosomes described herein are CD9⁺, CD10⁺, CD13⁺, CD29⁺, CD44⁺, CD49b⁺,CD49c⁺, CD55⁺, CD59⁺, CD63⁺, CD73⁺, CD81⁺, CD82⁺, CD90⁺, CD98⁺, CD105⁺,CD141⁺, CD142⁺, CD151⁺, CD164⁺, CD295⁺, CD200⁺, CD3⁻, CD11b⁻, CD14⁻,CD19⁻, CD33⁻, CD192⁻, HLA-A⁻, HLA-B⁻, HLA-C⁻, HLA-DR⁻, CD11c⁻ and CD34⁻.

In another embodiment, the placenta-derived adherent cell exosomesdescribed herein do not contain detectable levels of CD4, CD5, CD6, CD7,CD8, CD16, CD24, CD25, CD26, CD27, CD32, CD35, CD37, CD39, CD45, CD46,CD54, CD56, CD61, CD74, CD86, CD88, CD91, CD99, CD103, CD108, CD112,CD117, CD119, CD120a, CD123, CD126, CD130, CD134, CD138, CD140a, CD140b,CD144, CD146, CD147, CD152, CD163, CD183, CD184, CD186, CD191, CD194,CD196, CDw198, CD202b, CD220, CD221, CD235a, CD252, CD266, CD271, CD273,CD274, CD318, CD326, CD333, CD334, HLA-DP, HLA-DQ, HLA-G, IgD, IgM, NG2,PDGF, and/or SSEA-3.

In certain embodiments, the placenta-derived adherent cell exosomesdescribed herein are CD82⁺ and contain a low level of CD141 (i.e., areCD141(low)) as determined, e.g., by flow cytometry, for example, byFACS.

In certain embodiments, the placenta-derived adherent cell exosomesdescribed herein comprise a surface marker at a greater amount thanexosomes known in the art, as determinable by, e.g., FACS. In a specificembodiment, the placenta-derived adherent cell exosomes described hereincomprise a higher amount of CD49c, CD82, CD90, and/or CD142 than theamount of said marker(s) in a non placenta-derived adherent cellexosome. In a specific embodiment, the placenta-derived adherent cellexosomes described herein comprise a higher amount of CD49c, CD82, CD90,and/or CD142 than the amount of said marker(s) that is present inexosomes derived from either pre-adipocyte mesenchymal stem cells orchorionic villi mesenchymal stem cells (e.g., the chorionic villimesenchymal stem cells described in Salomon et al., 2013, PLOS ONE, 8:7,e68451). In a specific embodiment, the placenta-derived adherent cellexosomes described herein comprise a higher amount of CD49c, CD73, CD82,CD90, and/or CD142 than the amount of said marker(s) that is present inexosomes derived from pre-adipocyte mesenchymal stem cells.

In a specific embodiment, the placenta-derived adherent cell exosomesdescribed herein comprise a lower amount of CD164 than the amount ofsaid marker(s) in a non placenta-derived adherent cell exosome. In aspecific embodiment, the placenta-derived adherent cell exosomesdescribed herein comprise a lower amount of CD164 than the amount ofsaid marker(s) that is present in exosomes derived from pre-adipocytemesenchymal stem cells or chorionic villi mesenchymal stem cells (e.g.,the chorionic villi mesenchymal stem cells described in Salomon et al.,2013, PLOS ONE, 8:7, e68451). In a specific embodiment, theplacenta-derived adherent cell exosomes described herein comprise CD164at a level two-fold, three-fold, four-fold, five-fold, six-fold,seven-fold, eight-fold, nine-fold, or ten-fold lower than exosomesderived from chorionic villi mesenchymal stem cells (e.g., the chorionicvilli mesenchymal stem cells described in Salomon et al., 2013, PLOSONE, 8:7, e68451) or pre-adipocyte mesenchymal stem cells, wherein CD164content is measured under the same experimental conditions (e.g., byflow cytometry, for example, by FACS), wherein the exosomes areharvested under like conditions, and wherein the cells of origin aregrown under like conditions. In another specific embodiment, theplacenta-derived adherent cell exosomes described herein comprise CD164at a level two-fold to three-fold, three-fold to four-fold, four-fold tofive-fold, five-fold to six-fold, six-fold to seven-fold, seven-fold toeight-fold, eight-fold to nine-fold or nine-fold to ten-fold lower thanexosomes derived from chorionic villi mesenchymal stem cells (e.g., thechorionic villi mesenchymal stem cells described in Salomon et al.,2013, PLOS ONE, 8:7, e68451) or pre-adipocyte mesenchymal stem cells,wherein CD164 content is measured under the same experimental conditions(e.g., by flow cytometry, for example, by FACS), wherein the exosomesare harvested under like conditions, and wherein the cells of origin aregrown under like conditions. In a specific embodiment, theplacenta-derived adherent cell exosomes described herein comprise CD164at a level two-fold, three-fold, four-fold, five-fold, six-fold,seven-fold, eight-fold, nine-fold, or ten-fold lower than exosomesderived from chorionic villi mesenchymal stem cells (e.g., the chorionicvilli mesenchymal stem cells described in Salomon et al., 2013, PLOSONE, 8:7, e68451) or pre-adipocyte mesenchymal stem cells, wherein CD164content is measured under the same experimental conditions (e.g., byflow cytometry, for example, by FACS), and wherein the same numberand/or mass of exosomes is used for each sample. In another specificembodiment, the placenta-derived adherent cell exosomes described hereincomprise CD164 at a level two-fold to three-fold, three-fold tofour-fold, four-fold to five-fold, five-fold to six-fold, six-fold toseven-fold, seven-fold to eight-fold, eight-fold to nine-fold ornine-fold to ten-fold lower than exosomes derived from chorionic villimesenchymal stem cells (e.g., the chorionic villi mesenchymal stem cellsdescribed in Salomon et al., 2013, PLOS ONE, 8:7, e68451) orpre-adipocyte mesenchymal stem cells, wherein CD164 content is measuredunder the same experimental conditions (e.g., by flow cytometry, forexample, by FACS), and wherein the same number and/or mass of exosomesis used for each sample.

In another specific embodiment, the placenta-derived adherent cellexosomes described herein comprise CD49c at a level two-fold,three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold,nine-fold, or ten-fold higher than exosomes derived from chorionic villimesenchymal stem cells (e.g., the chorionic villi mesenchymal stem cellsdescribed in Salomon et al., 2013, PLOS ONE, 8:7, e68451) orpre-adipocyte mesenchymal stem cells, wherein CD49c content is measuredunder the same experimental conditions (e.g., by flow cytometry, forexample, by FACS), wherein the exosomes are harvested under likeconditions, and wherein the cells of origin are grown under likeconditions. In another specific embodiment, the placenta-derivedadherent cell exosomes described herein comprise CD49c at a leveltwo-fold to three-fold, three-fold to four-fold, four-fold to five-fold,five-fold to six-fold, six-fold to seven-fold, seven-fold to eight-fold,eight-fold to nine-fold or nine-fold to ten-fold higher than exosomesderived from chorionic villi mesenchymal stem cells (e.g., the chorionicvilli mesenchymal stem cells described in Salomon et al., 2013, PLOSONE, 8:7, e68451) or pre-adipocyte mesenchymal stem cells, wherein CD49ccontent is measured under the same experimental conditions (e.g., byflow cytometry, for example, by FACS), wherein the exosomes areharvested under like conditions, and wherein the cells of origin aregrown under like conditions. In another specific embodiment, theplacenta-derived adherent cell exosomes described herein comprise CD49cat a level two-fold, three-fold, four-fold, five-fold, six-fold,seven-fold, eight-fold, nine-fold, or ten-fold higher than exosomesderived from chorionic villi mesenchymal stem cells (e.g., the chorionicvilli mesenchymal stem cells described in Salomon et al., 2013, PLOSONE, 8:7, e68451) or pre-adipocyte mesenchymal stem cells, wherein CD49ccontent is measured under the same experimental conditions (e.g., byflow cytometry, for example, by FACS), and wherein the same numberand/or mass of exosomes is used for each sample. In another specificembodiment, the placenta-derived adherent cell exosomes described hereincomprise CD49c at a level two-fold to three-fold, three-fold tofour-fold, four-fold to five-fold, five-fold to six-fold, six-fold toseven-fold, seven-fold to eight-fold, eight-fold to nine-fold ornine-fold to ten-fold higher than exosomes derived from chorionic villimesenchymal stem cells (e.g., the chorionic villi mesenchymal stem cellsdescribed in Salomon et al., 2013, PLOS ONE, 8:7, e68451) orpre-adipocyte mesenchymal stem cells, wherein CD49c content is measuredunder the same experimental conditions (e.g., by flow cytometry, forexample, by FACS), and wherein the same number and/or mass of exosomesis used for each sample.

In another specific embodiment, the placenta-derived adherent cellexosomes described herein comprise CD73 at a level two-fold, three-fold,four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, orten-fold higher than exosomes derived from chorionic villi mesenchymalstem cells (e.g., the chorionic villi mesenchymal stem cells describedin Salomon et al., 2013, PLOS ONE, 8:7, e68451) or pre-adipocytemesenchymal stem cells, wherein CD73 content is measured under the sameexperimental conditions (e.g., by flow cytometry, for example, by FACS),wherein the exosomes are harvested under like conditions, and whereinthe cells of origin are grown under like conditions. In another specificembodiment, the placenta-derived adherent cell exosomes described hereincomprise CD73 at a level two-fold to three-fold, three-fold tofour-fold, four-fold to five-fold, five-fold to six-fold, six-fold toseven-fold, seven-fold to eight-fold, eight-fold to nine-fold ornine-fold to ten-fold higher than exosomes derived from chorionic villimesenchymal stem cells (e.g., the chorionic villi mesenchymal stem cellsdescribed in Salomon et al., 2013, PLOS ONE, 8:7, e68451) orpre-adipocyte mesenchymal stem cells, wherein CD73 content is measuredunder the same experimental conditions (e.g., by flow cytometry, forexample, by FACS), wherein the exosomes are harvested under likeconditions, and wherein the cells of origin are grown under likeconditions. In another specific embodiment, the placenta-derivedadherent cell exosomes described herein comprise CD73 at a leveltwo-fold, three-fold, four-fold, five-fold, six-fold, seven-fold,eight-fold, nine-fold, or ten-fold higher than exosomes derived fromchorionic villi mesenchymal stem cells (e.g., the chorionic villimesenchymal stem cells described in Salomon et al., 2013, PLOS ONE, 8:7,e68451) or pre-adipocyte mesenchymal stem cells, wherein CD73 content ismeasured under the same experimental conditions (e.g., by flowcytometry, for example, by FACS), and wherein the same number and/ormass of exosomes is used for each sample. In another specificembodiment, the placenta-derived adherent cell exosomes described hereincomprise CD73 at a level two-fold to three-fold, three-fold tofour-fold, four-fold to five-fold, five-fold to six-fold, six-fold toseven-fold, seven-fold to eight-fold, eight-fold to nine-fold ornine-fold to ten-fold higher than exosomes derived from chorionic villimesenchymal stem cells (e.g., the chorionic villi mesenchymal stem cellsdescribed in Salomon et al., 2013, PLOS ONE, 8:7, e68451) orpre-adipocyte mesenchymal stem cells, wherein CD73 content is measuredunder the same experimental conditions (e.g., by flow cytometry, forexample, by FACS), and wherein the same number and/or mass of exosomesis used for each sample.

In a specific embodiment, the placenta-derived adherent cell exosomesdescribed herein comprise CD82 at a level two-fold, three-fold,four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, orten-fold higher than exosomes derived from chorionic villi mesenchymalstem cells (e.g., the chorionic villi mesenchymal stem cells describedin Salomon et al., 2013, PLOS ONE, 8:7, e68451) or pre-adipocytemesenchymal stem cells, wherein CD82 content is measured under the sameexperimental conditions (e.g., by flow cytometry, for example, by FACS),wherein the exosomes are harvested under like conditions, and whereinthe cells of origin are grown under like conditions. In another specificembodiment, the placenta-derived adherent cell exosomes described hereincomprise CD82 at a level two-fold to three-fold, three-fold tofour-fold, four-fold to five-fold, five-fold to six-fold, six-fold toseven-fold, seven-fold to eight-fold, eight-fold to nine-fold ornine-fold to ten-fold higher than exosomes derived from chorionic villimesenchymal stem cells (e.g., the chorionic villi mesenchymal stem cellsdescribed in Salomon et al., 2013, PLOS ONE, 8:7, e68451) orpre-adipocyte mesenchymal stem cells, wherein CD82 content is measuredunder the same experimental conditions (e.g., by flow cytometry, forexample, by FACS), wherein the exosomes are harvested under likeconditions, and wherein the cells of origin are grown under likeconditions. In a specific embodiment, the placenta-derived adherent cellexosomes described herein comprise CD82 at a level two-fold, three-fold,four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, orten-fold higher than exosomes derived from chorionic villi mesenchymalstem cells (e.g., the chorionic villi mesenchymal stem cells describedin Salomon et al., 2013, PLOS ONE, 8:7, e68451) or pre-adipocytemesenchymal stem cells, wherein CD82 content is measured under the sameexperimental conditions (e.g., by flow cytometry, for example, by FACS),and wherein the same number and/or mass of exosomes is used for eachsample. In another specific embodiment, the placenta-derived adherentcell exosomes described herein comprise CD82 at a level two-fold tothree-fold, three-fold to four-fold, four-fold to five-fold, five-foldto six-fold, six-fold to seven-fold, seven-fold to eight-fold,eight-fold to nine-fold or nine-fold to ten-fold higher than exosomesderived from chorionic villi mesenchymal stem cells (e.g., the chorionicvilli mesenchymal stem cells described in Salomon et al., 2013, PLOSONE, 8:7, e68451) or pre-adipocyte mesenchymal stem cells, wherein CD82content is measured under the same experimental conditions (e.g., byflow cytometry, for example, by FACS), and wherein the same numberand/or mass of exosomes is used for each sample.

In a specific embodiment, the placenta-derived adherent cell exosomesdescribed herein comprise CD90 at a level two-fold, three-fold,four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, orten-fold higher than exosomes derived from chorionic villi mesenchymalstem cells (e.g., the chorionic villi mesenchymal stem cells describedin Salomon et al., 2013, PLOS ONE, 8:7, e68451) or pre-adipocytemesenchymal stem cells, wherein CD90 content is measured under the sameexperimental conditions (e.g., by flow cytometry, for example, by FACS),wherein the exosomes are harvested under like conditions, and whereinthe cells of origin are grown under like conditions. In another specificembodiment, the placenta-derived adherent cell exosomes described hereincomprise CD90 at a level two-fold to three-fold, three-fold tofour-fold, four-fold to five-fold, five-fold to six-fold, six-fold toseven-fold, seven-fold to eight-fold, eight-fold to nine-fold ornine-fold to ten-fold higher than exosomes derived from chorionic villimesenchymal stem cells (e.g., the chorionic villi mesenchymal stem cellsdescribed in Salomon et al., 2013, PLOS ONE, 8:7, e68451) orpre-adipocyte mesenchymal stem cells, wherein CD90 content is measuredunder the same experimental conditions (e.g., by flow cytometry, forexample, by FACS), wherein the exosomes are harvested under likeconditions, and wherein the cells of origin are grown under likeconditions. In a specific embodiment, the placenta-derived adherent cellexosomes described herein comprise CD90 at a level two-fold, three-fold,four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, orten-fold higher than exosomes derived from chorionic villi mesenchymalstem cells (e.g., the chorionic villi mesenchymal stem cells describedin Salomon et al., 2013, PLOS ONE, 8:7, e68451) or pre-adipocytemesenchymal stem cells, wherein CD90 content is measured under the sameexperimental conditions (e.g., by flow cytometry, for example, by FACS),and wherein the same number and/or mass of exosomes is used for eachsample. In another specific embodiment, the placenta-derived adherentcell exosomes described herein comprise CD90 at a level two-fold tothree-fold, three-fold to four-fold, four-fold to five-fold, five-foldto six-fold, six-fold to seven-fold, seven-fold to eight-fold,eight-fold to nine-fold or nine-fold to ten-fold higher than exosomesderived from chorionic villi mesenchymal stem cells (e.g., the chorionicvilli mesenchymal stem cells described in Salomon et al., 2013, PLOSONE, 8:7, e68451) or pre-adipocyte mesenchymal stem cells, wherein CD90content is measured under the same experimental conditions (e.g., byflow cytometry, for example, by FACS), and wherein the same numberand/or mass of exosomes is used for each sample.

In another specific embodiment, placenta-derived adherent cell exosomesdescribed herein comprise CD142 at a level two-fold, three-fold,four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, orten-fold higher than exosomes derived from chorionic villi mesenchymalstem cells (e.g., the chorionic villi mesenchymal stem cells describedin Salomon et al., 2013, PLOS ONE, 8:7, e68451) or pre-adipocytemesenchymal stem cells, wherein CD142 content is measured under the sameexperimental conditions (e.g., by flow cytometry, for example, by FACS),wherein the exosomes are harvested under like conditions, and whereinthe cells of origin are grown under like conditions. In another specificembodiment, the placenta-derived adherent cell exosomes described hereincomprise CD142 at a level two-fold to three-fold, three-fold tofour-fold, four-fold to five-fold, five-fold to six-fold, six-fold toseven-fold, seven-fold to eight-fold, eight-fold to nine-fold ornine-fold to ten-fold higher than exosomes derived from chorionic villimesenchymal stem cells (e.g., the chorionic villi mesenchymal stem cellsdescribed in Salomon et al., 2013, PLOS ONE, 8:7, e68451) orpre-adipocyte mesenchymal stem cells, wherein CD142 content is measuredunder the same experimental conditions (e.g., by flow cytometry, forexample, by FACS), wherein the exosomes are harvested under likeconditions, and wherein the cells of origin are grown under likeconditions. In another specific embodiment, placenta-derived adherentcell exosomes described herein comprise CD142 at a level two-fold,three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold,nine-fold, or ten-fold higher than exosomes derived from chorionic villimesenchymal stem cells (e.g., the chorionic villi mesenchymal stem cellsdescribed in Salomon et al., 2013, PLOS ONE, 8:7, e68451) orpre-adipocyte mesenchymal stem cells, wherein CD142 content is measuredunder the same experimental conditions (e.g., by flow cytometry, forexample, by FACS), and wherein the same number and/or mass of exosomesis used for each sample. In another specific embodiment, theplacenta-derived adherent cell exosomes described herein comprise CD142at a level two-fold to three-fold, three-fold to four-fold, four-fold tofive-fold, five-fold to six-fold, six-fold to seven-fold, seven-fold toeight-fold, eight-fold to nine-fold or nine-fold to ten-fold higher thanexosomes derived from chorionic villi mesenchymal stem cells (e.g., thechorionic villi mesenchymal stem cells described in Salomon et al.,2013, PLOS ONE, 8:7, e68451) or pre-adipocyte mesenchymal stem cells,wherein CD142 content is measured under the same experimental conditions(e.g., by flow cytometry, for example, by FACS), and wherein the samenumber and/or mass of exosomes is used for each sample.

In another specific embodiment, the placenta-derived adherent cellexosomes described herein comprise CD49c, CD90, CD142 and CD82 at alevel two-fold, three-fold, four-fold, five-fold, six-fold, seven-fold,eight-fold, nine-fold, or ten-fold higher than exosomes derived fromchorionic villi mesenchymal stem cells (e.g., the chorionic villimesenchymal stem cells described in Salomon et al., 2013, PLOS ONE, 8:7,e68451) or pre-adipocyte mesenchymal stem cells, wherein CD49c, CD90,CD142 and CD82 content is measured under the same experimentalconditions (e.g., by flow cytometry, for example, by FACS), wherein theexosomes are harvested under like conditions, and wherein the cells oforigin are grown under like conditions. In another specific embodiment,the placenta-derived adherent cell exosomes described herein compriseCD49c, CD90, CD142 and CD82 at a level two-fold to three-fold,three-fold to four-fold, four-fold to five-fold, five-fold to six-fold,six-fold to seven-fold, seven-fold to eight-fold, eight-fold tonine-fold or nine-fold to ten-fold higher than exosomes derived fromchorionic villi mesenchymal stem cells (e.g., the chorionic villimesenchymal stem cells described in Salomon et al., 2013, PLOS ONE, 8:7,e68451) or pre-adipocyte mesenchymal stem cells, wherein CD49c, CD90,CD142 and CD82 content is measured under the same experimentalconditions (e.g., by flow cytometry, for example, by FACS), wherein theexosomes are harvested under like conditions, and wherein the cells oforigin are grown under like conditions. In another specific embodiment,the placenta-derived adherent cell exosomes described herein compriseCD49c, CD90, CD142 and CD82 at a level two-fold, three-fold, four-fold,five-fold, six-fold, seven-fold, eight-fold, nine-fold, or ten-foldhigher than exosomes derived from chorionic villi mesenchymal stem cells(e.g., the chorionic villi mesenchymal stem cells described in Salomonet al., 2013, PLOS ONE, 8:7, e68451) or pre-adipocyte mesenchymal stemcells, wherein CD49c, CD90, CD142 and CD82 content is measured under thesame experimental conditions (e.g., by flow cytometry, for example, byFACS), and wherein the same number and/or mass of exosomes is used foreach sample. In another specific embodiment, the placenta-derivedadherent cell exosomes described herein comprise CD49c, CD90, CD142 andCD82 at a level two-fold to three-fold, three-fold to four-fold,four-fold to five-fold, five-fold to six-fold, six-fold to seven-fold,seven-fold to eight-fold, eight-fold to nine-fold or nine-fold toten-fold higher than exosomes derived from chorionic villi mesenchymalstem cells (e.g., the chorionic villi mesenchymal stem cells describedin Salomon et al., 2013, PLOS ONE, 8:7, e68451) or pre-adipocytemesenchymal stem cells, wherein CD49c, CD90, CD142 and CD82 content ismeasured under the same experimental conditions (e.g., by flowcytometry, for example, by FACS), and wherein the same number and/ormass of exosomes is used for each sample.

In addition to the exosomal markers described herein, placenta-derivedadherent cell exosomes described herein may additionally comprisecertain other exosomal markers. For example, in specific embodiments,the placenta-derived adherent cell exosomes described herein compriseexosomal surface markers FLOT1, ALIX, ANXA5, and TSG101 in addition toany marker combination described herein. In another embodiment,disclosed herein are compositions of placenta-derived adherent cellexosomes containing any combination of the above-referenced surfacemarkers.

5.1.1.2. MicroRNAs

In certain embodiments, the placenta-derived adherent cell exosomesdescribed herein contain one or more nucleic acid molecules, e.g., oneor more non-coding RNA molecules. In a specific embodiment, theplacenta-derived adherent cell exosomes provided herein comprisemicroRNAs (miRNAs). Generally, miRNAs are 17-22 nucleotide (nt)non-coding RNAs that modulate a broad range of cellular functions andare implicated in diseases and metabolic regulation. Methods ofdetecting miRNA levels are well known in the art, e.g., real-time PCR(RT-PCR), for example, Taqman assay. In other specific embodiments, theplacenta-derived adherent cell exosomes contain miRNAs that, whensubjected to RT-PCR and compared to a control RNA, are contained at alevel 1.1, 2, 5, 10, 100, 500, 600, 700, 800, 900, 1000, 1100 or 1200times higher than the same miRNA in chorionic villi mesenchymal stemcells, or between 1.1 and 2, 2 and 5, 2 and 10, 5 and 10, 10 and 100,100 and 500, 500 and 600, 600 and 700, 700 and 800, 800 and 900, 900 and1000, 1000 and 1100, or 1100 and 1200 times higher than the same miRNAin chorionic villi mesenchymal stem cells.

In certain embodiments, the placenta-derived adherent cell exosomesdescribed herein contain detectable levels of one, two, three, four ormore of the following miRNAs: miR-218-5p, miR-133b, miR-422a, miR-564,miR-16-5p, let-7a, miR-92, miR-142-3p, miR-451, miR-124-5p, miR-223-3p,miR-630, miR-296-5p, let-7b-3p and let-7d-3p. In one embodiment, theplacenta-derived adherent cell exosomes described herein comprisedetectable levels of miR-218-5p. In another embodiment, theplacenta-derived adherent cell exosomes described herein comprisedetectable levels of miR-133b. In another embodiment, theplacenta-derived adherent cell exosomes described herein comprisedetectable levels of miR-422a. In another embodiment, theplacenta-derived adherent cell exosomes described herein comprisedetectable levels of miR-564. In another embodiment, theplacenta-derived adherent cell exosomes described herein comprisedetectable levels of miR-16-5p. In another embodiment, theplacenta-derived adherent cell exosomes described herein comprisedetectable levels of let-7a. In another embodiment, the placenta-derivedadherent cell exosomes described herein comprise detectable levels ofmiR-92. In another embodiment, the placenta-derived adherent cellexosomes described herein comprise detectable levels of miR-142-3p. Inanother embodiment, the placenta-derived adherent cell exosomesdescribed herein comprise detectable levels of miR-451. In anotherembodiment, the placenta-derived adherent cell exosomes described hereincomprise detectable levels of miR-124-5p. In another embodiment, theplacenta-derived adherent cell exosomes described herein comprisedetectable levels of miR-223-3p. In another embodiment, theplacenta-derived adherent cell exosomes described herein comprisedetectable levels of miR-630. In another embodiment, theplacenta-derived adherent cell exosomes described herein comprisedetectable levels of miR-296-5p. In another embodiment, theplacenta-derived adherent cell exosomes described herein comprisedetectable levels of let-7b-3p. In another embodiment, theplacenta-derived adherent cell exosomes described herein comprisedetectable levels of let-7d-3p.

In one embodiment, the placenta-derived adherent cell exosomes describedherein comprise detectable levels of miR-218-5p and additionallycomprise detectable levels of one or more of the miRNAs selected fromthe group of miR-133b, miR-422a, miR-564, miR-16-5p, let-7a, miR-92,miR-142-3p, miR-451, miR-124-5p, miR-223-3p, miR-630, miR-296-5p,let-7b-3p and let-7d-3p.

In another embodiment, the placenta-derived adherent cell exosomesdescribed herein comprise detectable levels of miR-133b and additionallycomprise detectable levels of one or more of the miRNAs selected fromthe group of miR-218-5p, miR-422a, miR-564, miR-16-5p, let-7a, miR-92,miR-142-3p, miR-451, miR-124-5p, miR-223-3p, miR-630, miR-296-5p,let-7b-3p and let-7d-3p.

In another embodiment, the placenta-derived adherent cell exosomesdescribed herein comprise detectable levels of miR-422a and additionallycomprise detectable levels of one or more of the miRNAs selected fromthe group of miR-218-5p, miR-133b, miR-564, miR-16-5p, let-7a, miR-92,miR-142-3p, miR-451, miR-124-5p, miR-223-3p, miR-630, miR-296-5p,let-7b-3p and let-7d-3p.

In another embodiment, the placenta-derived adherent cell exosomesdescribed herein comprise detectable levels of miR-564 and additionallycomprise detectable levels of one or more of the miRNAs selected fromthe group of miR-218-5p, miR-133b, miR-422a, miR-16-5p, let-7a, miR-92,miR-142-3p, miR-451, miR-124-5p, miR-223-3p, miR-630, miR-296-5p,let-7b-3p and let-7d-3p.

In another embodiment, the placenta-derived adherent cell exosomesdescribed herein comprise detectable levels of miR-16-5p andadditionally comprise detectable levels of one or more of the miRNAsselected from the group of miR-218-5p, miR-133b, miR-422a, miR-564,let-7a, miR-92, miR-142-3p, miR-451, miR-124-5p, miR-223-3p, miR-630,miR-296-5p, let-7b-3p and let-7d-3p.

In another embodiment, the placenta-derived adherent cell exosomesdescribed herein comprise detectable levels of let-7a and additionallycomprise detectable levels of one or more of the miRNAs selected fromthe group of miR-218-5p, miR-133b, miR-422a, miR-564, miR-16-5p, miR-92,miR-142-3p, miR-451, miR-124-5p, miR-223-3p, miR-630, miR-296-5p,let-7b-3p and let-7d-3p.

In another embodiment, the placenta-derived adherent cell exosomesdescribed herein comprise detectable levels of miR-92 and additionallycomprise detectable levels of one or more of the miRNAs selected fromthe group of miR-218-5p, miR-133b, miR-422a, miR-564, miR-16-5p, let-7a,miR-142-3p, miR-451, miR-124-5p, miR-223-3p, miR-630, miR-296-5p,let-7b-3p and let-7d-3p.

In another embodiment, the placenta-derived adherent cell exosomesdescribed herein comprise detectable levels of miR-142-3p andadditionally comprise detectable levels of one or more of the miRNAsselected from the group of miR-218-5p, miR-133b, miR-422a, miR-564,miR-16-5p, let-7a, miR-92, miR-451, miR-124-5p, miR-223-3p, miR-630,miR-296-5p, let-7b-3p and let-7d-3p.

In another embodiment, the placenta-derived adherent cell exosomesdescribed herein comprise detectable levels of miR-451 and additionallycomprise detectable levels of one or more of the miRNAs selected fromthe group of miR-218-5p, miR-133b, miR-422a, miR-564, miR-16-5p, let-7a,miR-92, miR-142-3p, miR-124-5p, miR-223-3p, miR-630, miR-296-5p,let-7b-3p and let-7d-3p.

In another embodiment, the placenta-derived adherent cell exosomesdescribed herein comprise detectable levels of miR-124-5p andadditionally comprise detectable levels of one or more of the miRNAsselected from the group of miR-218-5p, miR-133b, miR-422a, miR-564,miR-16-5p, let-7a, miR-92, miR-142-3p, miR-451, miR-223-3p, miR-630,miR-296-5p, let-7b-3p and let-7d-3p.

In another embodiment, the placenta-derived adherent cell exosomesdescribed herein comprise detectable levels of miR-223-3p andadditionally comprise detectable levels of one or more of the miRNAsselected from the group of miR-218-5p, miR-133b, miR-422a, miR-564,miR-16-5p, let-7a, miR-92, miR-142-3p, miR-451, miR-124-5p, miR-630,miR-296-5p, let-7b-3p and let-7d-3p.

In another embodiment, the placenta-derived adherent cell exosomesdescribed herein comprise detectable levels of miR-630 and additionallycomprise detectable levels of one or more of the miRNAs selected fromthe group of miR-218-5p, miR-133b, miR-422a, miR-564, miR-16-5p, let-7a,miR-92, miR-142-3p, miR-451, miR-124-5p, miR-223-3p, miR-296-5p,let-7b-3p and let-7d-3p.

In another embodiment, the placenta-derived adherent cell exosomesdescribed herein comprise detectable levels of miR-296-5p andadditionally comprise detectable levels of one or more of the miRNAsselected from the group of miR-218-5p, miR-133b, miR-422a, miR-564,miR-16-5p, let-7a, miR-92, miR-142-3p, miR-451, miR-124-5p, miR-223-3p,miR-630, let-7b-3p and let-7d-3p.

In another embodiment, the placenta-derived adherent cell exosomesdescribed herein comprise detectable levels of let-7b-3p andadditionally comprise detectable levels of one or more of the miRNAsselected from the group of miR-218-5p, miR-133b, miR-422a, miR-564,miR-16-5p, let-7a, miR-92, miR-142-3p, miR-451, miR-124-5p, miR-223-3p,miR-630, miR-296-5p and let-7d-3p.

In another embodiment, the placenta-derived adherent cell exosomesdescribed herein comprise detectable levels of let-7d-3p andadditionally comprise detectable levels of one or more of the miRNAsselected from the group of miR-218-5p, miR-133b, miR-422a, miR-564,miR-16-5p, let-7a, miR-92, miR-142-3p, miR-451, miR-124-5p, miR-223-3p,miR-630, miR-296-5p and let-7b-3p.

In a specific embodiment, the placenta-derived adherent cell exosomesdescribed herein comprise miR-218-5p, miR-133b, miR-422a, miR-564,miR-16-5p, let-7a, miR-92, miR-142-3p, miR-451, miR-124-5p, miR-223-3p,miR-630, miR-296-5p, let-7b-3p and let-7d-3p.

In certain embodiments, the placenta-derived adherent cell exosomesdescribed herein can be distinguished from exosomes from other cells(e.g., chorionic villi mesenchymal stem cells, e.g., the chorionic villimesenchymal stem cells described in Salomon et al., 2013, PLOS ONE, 8:7,e68451) based on the miRNA they comprise.

In a specific embodiment, the placenta-derived adherent cell exosomesdescribed herein comprise miR-133b at a level at least two-fold higherwhen compared to exosomes from chorionic villi mesenchymal stem cells(e.g., the chorionic villi mesenchymal stem cells described in Salomonet al., 2013, PLOS ONE, 8:7, e68451) when the exosomes and the cellsfrom which they are derived are treated under like conditions.

In another embodiment, the placenta-derived adherent cell exosomesdescribed herein comprise miR-422a at a level at least two-fold higheras compared to exosomes from chorionic villi mesenchymal stem cells(e.g., the chorionic villi mesenchymal stem cells described in Salomonet al., 2013, PLOS ONE, 8:7, e68451) when the exosomes and the cellsfrom which they are derived are treated by like methods.

In another embodiment, the placenta-derived adherent cell exosomesdescribed herein comprise miR-16-5p at a level at least two-fold higheras compared to exosomes from chorionic villi mesenchymal stem cells(e.g., the chorionic villi mesenchymal stem cells described in Salomonet al., 2013, PLOS ONE, 8:7, e68451) when the exosomes and the cellsfrom which they are derived are treated by like methods.

In another embodiment, the placenta-derived adherent cell exosomesdescribed herein comprise let-7a at a level at least two-fold higher ascompared to exosomes from chorionic villi mesenchymal stem cells (e.g.,the chorionic villi mesenchymal stem cells described in Salomon et al.,2013, PLOS ONE, 8:7, e68451) when the exosomes and the cells from whichthey are derived are treated by like methods.

In another embodiment, the placenta-derived adherent cell exosomesdescribed herein comprise miR-92 at a level at least two-fold higher ascompared to exosomes from chorionic villi mesenchymal stem cells (e.g.,the chorionic villi mesenchymal stem cells described in Salomon et al.,2013, PLOS ONE, 8:7, e68451) when the exosomes and the cells from whichthey are derived are treated by like methods.

In another embodiment, the placenta-derived adherent cell exosomesdescribed herein comprise miR-142-3p at a level at least two-fold higheras compared to exosomes from chorionic villi mesenchymal stem cells(e.g., the chorionic villi mesenchymal stem cells described in Salomonet al., 2013, PLOS ONE, 8:7, e68451) when the exosomes and the cellsfrom which they are derived are treated by like methods.

In another embodiment, the placenta-derived adherent cell exosomesdescribed herein comprise miR-451 at a level at least two-fold higher ascompared to exosomes from chorionic villi mesenchymal stem cells (e.g.,the chorionic villi mesenchymal stem cells described in Salomon et al.,2013, PLOS ONE, 8:7, e68451) when the exosomes and the cells from whichthey are derived are treated by like methods.

In another embodiment, the placenta-derived adherent cell exosomesdescribed herein comprise miR-124-5p at a level at least two-fold higheras compared to exosomes from chorionic villi mesenchymal stem cells(e.g., the chorionic villi mesenchymal stem cells described in Salomonet al., 2013, PLOS ONE, 8:7, e68451) when the exosomes and the cellsfrom which they are derived are treated by like methods.

In another embodiment, the placenta-derived adherent cell exosomesdescribed herein comprise miR-223-3p at a level at least two-fold higheras compared to exosomes from chorionic villi mesenchymal stem cells(e.g., the chorionic villi mesenchymal stem cells described in Salomonet al., 2013, PLOS ONE, 8:7, e68451) when the exosomes and the cellsfrom which they are derived are treated by like methods.

In another embodiment, the placenta-derived adherent cell exosomesdescribed herein comprise miR-630 at a level at least two-fold higher ascompared to exosomes from chorionic villi mesenchymal stem cells (e.g.,the chorionic villi mesenchymal stem cells described in Salomon et al.,2013, PLOS ONE, 8:7, e68451) when the exosomes and the cells from whichthey are derived are treated by like methods.

In another embodiment, the placenta-derived adherent cell exosomesdescribed herein comprise miR-296-5p at a level at least two-fold higheras compared to exosomes from chorionic villi mesenchymal stem cells(e.g., the chorionic villi mesenchymal stem cells described in Salomonet al., 2013, PLOS ONE, 8:7, e68451) when the exosomes and the cellsfrom which they are derived are treated by like methods.

In another embodiment, the placenta-derived adherent cell exosomesdescribed herein comprise let-7b-3p at a level at least two-fold higheras compared to exosomes from chorionic villi mesenchymal stem cells(e.g., the chorionic villi mesenchymal stem cells described in Salomonet al., 2013, PLOS ONE, 8:7, e68451) when the exosomes and the cellsfrom which they are derived are treated by like methods.

In another embodiment, the placenta-derived adherent cell exosomesdescribed herein comprise let-7d-3p at a level at least two-fold higheras compared to exosomes from chorionic villi mesenchymal stem cells(e.g., the chorionic villi mesenchymal stem cells described in Salomonet al., 2013, PLOS ONE, 8:7, e68451) when the exosomes and the cellsfrom which they are derived are treated by like methods.

In one embodiment, the placenta-derived adherent cell exosomes describedherein comprise miR-133b and additionally one or more miRNA selectedfrom the group of miR-422a, miR-16-5p, let-7a, miR-92, miR-142-3p,miR-451, miR-223-3p, miR-630, miR-296-5p, let-7b-3p and let-7d-3p at alevel at least two-fold higher as compared to exosomes from chorionicvilli mesenchymal stem cells (e.g., the chorionic villi mesenchymal stemcells described in Salomon et al., 2013, PLOS ONE, 8:7, e68451) when theexosomes and the cells from which they are derived are treated by likemethods.

In one embodiment, the placenta-derived adherent cell exosomes describedherein comprise miR-422a and additionally one or more miRNA selectedfrom the group of miR-133b, miR-16-5p, let-7a, miR-92, miR-142-3p,miR-451, miR-223-3p, miR-630, miR-296-5p, let-7b-3p and let-7d-3p at alevel at least two-fold higher as compared to exosomes from chorionicvilli mesenchymal stem cells (e.g., the chorionic villi mesenchymal stemcells described in Salomon et al., 2013, PLOS ONE, 8:7, e68451) when theexosomes and the cells from which they are derived are treated by likemethods.

In one embodiment, the placenta-derived adherent cell exosomes describedherein comprise miR-16-5p and additionally one or more miRNA selectedfrom the group of miR-133b, miR-422a, let-7a, miR-92, miR-142-3p,miR-451, miR-223-3p, miR-630, miR-296-5p, let-7b-3p and let-7d-3p at alevel at least two-fold higher as compared to exosomes from chorionicvilli mesenchymal stem cells (e.g., the chorionic villi mesenchymal stemcells described in Salomon et al., 2013, PLOS ONE, 8:7, e68451) when theexosomes and the cells from which they are derived are treated by likemethods.

In one embodiment, the placenta-derived adherent cell exosomes describedherein comprise let-7a and additionally one or more miRNA selected fromthe group of miR-133b, miR-422a, miR-16-5p, miR-92, miR-142-3p, miR-451,miR-223-3p, miR-630, miR-296-5p, let-7b-3p and let-7d-3p at a level atleast two-fold higher as compared to exosomes from chorionic villimesenchymal stem cells (e.g., the chorionic villi mesenchymal stem cellsdescribed in Salomon et al., 2013, PLOS ONE, 8:7, e68451) when theexosomes and the cells from which they are derived are treated by likemethods.

In one embodiment, the placenta-derived adherent cell exosomes describedherein comprise miR-92 and additionally one or more miRNA selected fromthe group of miR-133b, miR-422a, miR-16-5p, let-7a, miR-142-3p, miR-451,miR-223-3p, miR-630, miR-296-5p, let-7b-3p and let-7d-3p at a level atleast two-fold higher as compared to exosomes from chorionic villimesenchymal stem cells (e.g., the chorionic villi mesenchymal stem cellsdescribed in Salomon et al., 2013, PLOS ONE, 8:7, e68451) when theexosomes and the cells from which they are derived are treated by likemethods.

In one embodiment, the placenta-derived adherent cell exosomes describedherein comprise miR-142-3p and additionally one or more miRNA selectedfrom the group of miR-133b, miR-422a, miR-16-5p, let-7a, miR-92,miR-451, miR-223-3p, miR-630, miR-296-5p, let-7b-3p and let-7d-3p at alevel at least two-fold higher as compared to exosomes from chorionicvilli mesenchymal stem cells (e.g., the chorionic villi mesenchymal stemcells described in Salomon et al., 2013, PLOS ONE, 8:7, e68451) when theexosomes and the cells from which they are derived are treated by likemethods.

In one embodiment, the placenta-derived adherent cell exosomes describedherein comprise miR-451 and additionally one or more miRNA selected fromthe group of miR-133b, miR-422a, miR-16-5p, let-7a, miR-92, miR-142-3p,miR-223-3p, miR-630, miR-296-5p, let-7b-3p and let-7d-3p at leasttwo-fold higher as compared to exosomes from chorionic villi mesenchymalstem cells (e.g., the chorionic villi mesenchymal stem cells describedin Salomon et al., 2013, PLOS ONE, 8:7, e68451) when the exosomes andthe cells from which they are derived are treated by like methods.

In one embodiment, the placenta-derived adherent cell exosomes describedherein comprise miR-223-3p and additionally one or more miRNA selectedfrom the group of miR-133b, miR-422a, miR-16-5p, let-7a, miR-92,miR-142-3p, miR-451, miR-630, miR-296-5p, let-7b-3p and let-7d-3p at alevel at least two-fold higher as compared to exosomes from chorionicvilli mesenchymal stem cells (e.g., the chorionic villi mesenchymal stemcells described in Salomon et al., 2013, PLOS ONE, 8:7, e68451) when theexosomes and the cells from which they are derived are treated by likemethods.

In one embodiment, the placenta-derived adherent cell exosomes describedherein comprise miR-630 and additionally one or more miRNA selected fromthe group of miR-133b, miR-422a, miR-16-5p, let-7a, miR-92, miR-142-3p,miR-451, miR-223-3p, miR-296-5p, let-7b-3p and let-7d-3p at a level atleast two-fold higher as compared to exosomes from chorionic villimesenchymal stem cells (e.g., the chorionic villi mesenchymal stem cellsdescribed in Salomon et al., 2013, PLOS ONE, 8:7, e68451) when theexosomes and the cells from which they are derived are treated by likemethods.

In one embodiment, the placenta-derived adherent cell exosomes describedherein comprise miR-296-5p and additionally one or more miRNA selectedfrom the group of miR-133b, miR-422a, miR-16-5p, let-7a, miR-92,miR-142-3p, miR-451, miR-223-3p, miR-630, let-7b-3p and let-7d-3p at alevel at least two-fold higher as compared to exosomes from chorionicvilli mesenchymal stem cells (e.g., the chorionic villi mesenchymal stemcells described in Salomon et al., 2013, PLOS ONE, 8:7, e68451) when theexosomes and the cells from which they are derived are treated by likemethods.

In one embodiment, the placenta-derived adherent cell exosomes describedherein comprise let-7b-3p and additionally one or more miRNA selectedfrom the group of miR-133b, miR-422a, miR-16-5p, let-7a, miR-92,miR-142-3p, miR-451, miR-223-3p, miR-630, miR-296-5p and let-7d-3p at alevel at least two-fold higher as compared to exosomes from chorionicvilli mesenchymal stem cells (e.g., the chorionic villi mesenchymal stemcells described in Salomon et al., 2013, PLOS ONE, 8:7, e68451) when theexosomes and the cells from which they are derived are treated by likemethods.

In one embodiment, the placenta-derived adherent cell exosomes describedherein comprise let-7d-3p and additionally one or more miRNA selectedfrom the group of miR-133b, miR-422a, miR-16-5p, let-7a, miR-92,miR-142-3p, miR-451, miR-223-3p, miR-630, miR-296-5p and let-7b-3p at alevel at least two-fold higher as compared to exosomes from chorionicvilli mesenchymal stem cells (e.g., the chorionic villi mesenchymal stemcells described in Salomon et al., 2013, PLOS ONE, 8:7, e68451) when theexosomes and the cells from which they are derived are treated by likemethods.

In another embodiment, the placenta-derived adherent cell exosomesdescribed herein comprise miR-133b, miR-422a, miR-16-5p, let-7a, miR-92,miR-142-3p, miR-451, miR-223-3p, miR-630, miR-296-5p, let-7b-3p andlet-7d-3p or a combination of any of the foregoing at a level at leasttwo-fold higher when compared to exosomes from chorionic villimesenchymal stem cells (e.g., the chorionic villi mesenchymal stem cellsdescribed in Salomon et al., 2013, PLOS ONE, 8:7, e68451) when theexosomes and the cells from which they are derived are treated by likeconditions.

5.1.1.3. Angiogenic Proteins

In certain embodiments, the placenta-derived adherent cell exosomesdescribed herein contain one or more proteins that promote angiogenesis.Methods of detecting angiogenic protein levels are well known in theart, e.g., using an ELISA-based method, for example, AngioSecretomeMilliplex (Millipore). In specific embodiments, the presence andrelative levels of angiogenic proteins associated with theplacenta-derived adherent cell exosomes described herein is determinedby the method described in Section 6.2.4.3, infra.

In one embodiment, the placenta-derived adherent cell exosomes describedherein contain one or more angiogenic proteins selected from the groupof Endoglin, Leptin, Angiopoietin-2, G-CSF, Follistatin, FGF-2, HGF,VEGF-A, IL-8 and EGF. In another embodiment, the placenta-derivedadherent cell exosomes described herein contain one or more angiogenicproteins selected from the group of Endoglin, Leptin, Angiopoietin-2,G-CSF, Follistatin, FGF-2, HGF, VEGF-A, IL-8 and EGF at a level higherthan culture medium lacking exosomes. In another embodiment, saidangiogenic proteins are present in culture medium at a concentration ofgreater than 10 pg/ml, 50 pg/ml, 100 pg/ml, 500 pg/ml, 1,000 pg/ml,2,000 pg/ml, 4,000 pg/ml, 6,000 pg/ml, 8,000 pg/ml, 10,000 pg/ml, 12,000pg/ml, 14,000 pg/ml, 16,000 pg/ml, 18,000 pg/ml, 20,000 pg/ml, 25,000pg/ml, 30,000 pg/ml, 35,000 pg/ml, 40,000 pg/ml, 45,000 pg/ml, or 50,000pg/ml or higher. In another embodiment said angiogenic proteins arepresent in culture medium at a concentration of 10 pg/ml to 50 pg/ml, 50pg/ml to 100 pg/ml, 100 pg/ml to 500 pg/ml, 500 pg/ml to 1,000 pg/ml,1,000 pg/ml to 2,000 pg/ml, 2,000 pg/ml to 4,000 pg/ml, 4,000 pg/ml to6,000 pg/ml, 6,000 pg/ml to 8,000 pg/ml, 8,000 pg/ml to 10,000 pg/ml,10,000 pg/ml 12,000 pg/ml, 12,000 pg/ml to 14,000 pg/ml, 14,000 pg/ml to16,000 pg/ml, 16,000 pg/ml to 18,000 pg/ml, 18,000 pg/ml to 20,000pg/ml, 20,000 pg/ml to 25,000 pg/ml, 25,000 pg/ml to 30,000 pg/ml,30,000 pg/ml to 35,000 pg/ml, 35,000 pg/ml to 40,000 pg/ml, 40,000 pg/ml45,000 pg/ml, or 45,000 pg/ml to 50,000 pg/ml.

5.1.2. Exosome Size and Distinguishing Characteristics

In certain embodiments, the placenta-derived adherent cell exosomesdescribed herein are about 50 nm to 150 nm in diameter. In certainembodiments, the placenta-derived adherent cell exosomes describedherein are about 70 nm to 150 nm in diameter, about 100 nm to 150 nm indiameter, or about 130 nm to 145 nm in diameter, as determined bymethods known in the art (e.g., electron microscopy and NanoparticleTracking Assay). In certain embodiments, the placenta-derived adherentcell exosomes described herein can be distinguished from cellular and/ornon-cellular debris on the basis of their size. In certain embodiments,the exosomes described herein can be distinguished by their shape, forexample, the exosomes described herein are cup-shaped when viewed byimaging techniques known in the art (e.g., electron microscopy).

In certain embodiments, the placenta-derived adherent cell exosomesdescribed herein can be identified on the basis of their molecularcomposition (e.g., surface marker proteins or miRNA molecules). SeeSection 5.1.1. In certain embodiments, the placenta-derived adherentcell exosomes described herein can be distinguished from other exosomes(i.e., non-placenta-derived adherent cell exosomes) based theirmolecular profile (e.g., the surface marker proteins and/or miRNAmolecules they comprise). See Section 5.1.1.

5.1.3. Placenta-Derived Adherent Cells

The placenta-derived adherent cell exosomes described herein are derivedfrom placenta-derived adherent cells (e.g., the placenta-derivedadherent cells disclosed in U.S. Pat. Nos. 7,468,276; 8,057,788 and8,202,703, the disclosures of which are hereby incorporated by referencein their entireties).

In a specific embodiment, the placenta-derived adherent cell exosomesdescribed herein are derived from CD34⁻, CD10⁺, CD105⁺ and CD200⁺placenta-derived adherent cells.

In another specific embodiment, the placenta-derived adherent cellexosomes described herein are derived from placenta-derived adherentcells that express one or more genes at a level at least two-fold higherthan bone marrow-derived mesenchymal stem cells, wherein said genes areselected from the group consisting of ACTG2, ADARB1, AMIGO2, ARTS-1,B4GALT6, BCHE, Cllorf9, CD200, COL4A1, COL4A2, CPA4, DMD, DSC3, DSG2,ELOVL2, F2RL1, FIJ10781, GATA6, GPR126, GPRC5B, HLA-G, ICAM1, IER3,IGFBP7, IL1A, IL6, IL18, KRT18, KRT8, LIPG, LRAP, MATN2, MEST, NFE2L3,NUAK1, PCDH7, PDLIM3, PJP2, RTN1, SERPINB9, ST3GAL6, ST6GALNAC5,SLC12A8, TCF21, TGFB2, VTN, and ZC3H12A. In another embodiment, theplacenta-derived adherent cells from which the exosomes described hereinare derived express one or more genes at a level at least two-foldhigher than bone marrow-derived mesenchymal stem cells, wherein said oneor more genes are one or more of LOVL2, ST3GAL6, ST6GALNAC5, and/orSLC12A8. In another embodiment, the placenta-derived adherent cells fromwhich the exosomes provided herein are derived express one or more genesat a level at least two-fold higher than bone marrow-derived mesenchymalstem cells, wherein said one or more genes are one or more of CPA4,TCF21, VTN, B4GALT6, F1110781, and/or NUAK1.

In certain embodiments, the placenta-derived adherent cell exosomesdescribed herein are obtained from placenta-derived adherent cells grownin culture, e.g., placenta-derived adherent cells that have adhered to atissue culture substrate, or from placenta-derived adherent cells thathave administered to a living organism (e.g., a mammal, e.g., a human).In a specific embodiment, the cells are cultured in the presence ofserum. In another specific embodiment, the cells are cultured in theabsence of serum.

In one embodiment, the placenta-derived adherent cells from which theplacenta-derived adherent cell exosomes provided herein are derived aresubstantially fetal in origin, e.g., the placenta-derived adherent cellsare about or at least 90%, 95%, 99% or 100% fetal in origin. In anotherembodiment, the placenta-derived adherent cells from which theplacenta-derived adherent cell exosomes provided herein are derived areof mixed origin, e.g., the placenta-derived adherent cells are bothfetal and maternal in origin.

In certain embodiments, the placenta-derived adherent cells from whichthe placenta-derived adherent cell exosomes provided herein are derivedhave been passaged prior to isolation of exosomes from theplacenta-derived adherent cells. In a specific embodiment, theplacenta-derived adherent cells from which the placenta-derived adherentcell exosomes provided herein are derived have been passaged about or atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, or 20 times priorto isolation of exosomes from the placenta-derived adherent cells. In aspecific embodiment, the exosomes provided herein are collected fromplacenta-derived adherent cells that have been cultured in vitro for 6passages. In certain embodiments, the exosomes provided herein arecollected from placenta-derived adherent cells plated at passage 6, andcollected from culture supernatant at passage 7.

In certain embodiments, the placenta-derived adherent cells from whichthe placenta-derived adherent cell exosomes provided herein are derivedhave been expanded prior to isolation of exosomes from theplacenta-derived adherent cells. In a specific embodiment, theplacenta-derived adherent cells from which the placenta-derived adherentcell exosomes provided herein are derived have been expanded for 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34,36, 38 or 40 population doublings, or more, prior to isolation ofexosomes from the placenta-derived adherent cells.

In a specific embodiment, the placenta-derived adherent cells from whichthe placenta-derived adherent cell exosomes provided herein are derivedare not chorionic villi mesenchymal stem cells and/or do not comprisechorionic villi mesenchymal stem cells, e.g., the placenta-derivedadherent cells from which the placenta-derived adherent cell exosomesprovided herein are derived are not the chorionic villi mesenchymal stemcells described in Salomon et al., 2013, PLOS ONE, 8:7, e68451. Inanother embodiment, the source from which the placenta-derived adherentcells are derived does not contain chorionic villi.

5.2. Methods of Isolating Placenta-Derived Adherent Cell Exosomes

5.2.1. Methods of Obtaining Exosomes from Medium

The placenta-derived adherent cell exosomes described herein can beproduced from, e.g., the placenta-derived adherent cells described inSection 5.1.3. In certain embodiments, the culture of suchplacenta-derived adherent cells results in the deposition of exosomesinto the culture medium, wherein said exosomes may be isolated from theculture medium using techniques described herein and known in the art.The isolation of placenta-derived adherent cell exosomes describedherein may be accomplished based on specific surface marker moleculesassociated with the placenta-derived adherent cell exosomes, using sizefiltration, using density-dependent centrifugation, and by using othermethods known in the art. In certain embodiments, isolation of theplacenta-derived adherent cell exosomes described herein can combine twoor more techniques to substantially purify exosome populations fromcontaminating cellular and non-cellular material present in culturemedium.

5.2.1.1. Isolation of Exosomes by Ultracentrifugation

In a specific embodiment, the placenta-derived adherent cell exosomesdescribed herein are separated from other components of cell culturemedium using ultracentrifugation. In a specific embodiment, culturemedium collected from cultures of placenta-derived adherent cells isisolated and centrifuged at high speed (e.g., 100,000×g) for a timesufficient to separate placenta-derived adherent cell exosomes fromcellular and non-cellular materials present in the culture medium. In aspecific embodiment, placenta-derived adherent cells are cultured in thepresence of serum (e.g., fetal bovine serum) prior to collecting culturemedium for the isolation of placenta-derived adherent cell exosomes. Inanother specific embodiment, placenta-derived adherent cells arecultured in the absence of serum (e.g., fetal bovine serum) prior tocollecting culture medium for the isolation of placenta-derived adherentcell exosomes. In one embodiment, culture medium collected from culturesof placenta-derived adherent cells is isolated and centrifuged accordingto the method of Example 1, infra.

5.2.1.2. Isolation of exosomes by fluorescence-activated cell sorting

The placenta-derived adherent cell exosomes described herein can beisolated and/or purified on the basis of containing one or more surfacemarker proteins and/or on the basis of lacking one or more surfacemarker proteins. Specifically, the placenta-derived adherent cellexosomes described herein can be substantially isolated fromnon-placenta-derived adherent cell exosomes, or placenta-derivedadherent cell exosomes that do not contain the surface markers ofinterest. For example, one subpopulation of placenta-derived adherentcell exosomes containing a specific combination of surface markers canbe isolated from one or more additional populations of exosomes (e.g.,placenta-derived adherent cell exosomes) that contain a unique orsubstantially different combination of surface markers. Accordingly, theplacenta-derived adherent cell exosomes described herein can be isolatedand/or purified on the basis of any combination of the surface markersdescribed herein. See Section 5.1.1.

In one embodiment, fluorescence-activated cell sorting (FACS) is used toseparate and/or purify the placenta-derived adherent cell exosomesdescribed herein using antibodies specific to cell surface markerspresent on the exosomes. Methods of using FACS to isolate cells andexosomes are known in the art.

In one embodiment, the method of purifying/isolating theplacenta-derived adherent cell exosomes described herein comprisescontacting a population of exosomes described herein with a solidsubstrate (e.g., latex bead) for a time and under conditions sufficientto allow for the creation of a complex between said exosomes and saidsubstrate (e.g., by passive adsorption). In another specific embodiment,a population of exosomes contacted with a solid substrate (e.g., latexbeads) for a time and under conditions sufficient to allow for thecreation of a complex between said exosomes and said substrate (e.g., bypassive adsorption) is separated according to a method described herein(e.g., a FACS method described herein).

In another embodiment, the placenta-derived adherent cell exosomesdescribed herein can be purified and/or isolated using a two-part“sandwich” detection technique. In a particular embodiment, the exosomesin a partially purified or unpurified population are first contactedwith a substrate, e.g., a population of beads (e.g., a population oflatex beads), that are coated with an antibody specific for a proteinthat is present on the surface of the placenta-derived adherent cellexosomes described herein. In a specific embodiment, an antibody thatspecifically recognizes CD63 is used to isolate the placenta-derivedadherent cell exosomes described herein using such a method. In anotherspecific embodiment, an antibody that specifically recognizes a memberof the MHC class II molecule family is used to isolate theplacenta-derived adherent cell exosomes described herein using such amethod. After the first contacting step, the exosomes bound to thesubstrate are contacted by a second molecule specific to a surfacemarker protein that is present on the surface of the placenta-derivedadherent cell exosomes described herein. In a specific embodiment, thesurface molecule is a tetraspanin and said antibody is afluorescently-labeled antibody. In another specific embodiment, thesurface molecule is any one of FLOT1, ALIX, ANXA5, TSG101, Annexin V,CD3, CD9, CD10, CD11b, CD13, CD14, CD19, CD29, CD33, CD44, CD46, CD47,CD49b, CD49c, CD55, CD59, CD63, CD73, CD81, CD90, CD98, CD105, CD142,CD147, CD151, CD164, CD166, CD192, HLA-A, HLA-B, HLA-C, HLA-DR, CD11c,CD34, CD49d or CD200.

5.2.1.3. Isolation of Exosomes Using Size-Exclusion Chromatography

The placenta-derived adherent cell exosomes described herein can beisolated and/or purified from common contaminants present in culturemedium, including thyroglobulin dimers, thyroglobulin monomers,Immunglobulins (e.g., IgG), bovine serum albumin (BSA), myoglobulin, anduracil (see Section 6.3). In one embodiment, the placenta-derivedadherent cell exosomes described herein are distinguished in a samplefrom common culture medium contaminants (e.g., Thyroglobulin, IgG, BSA,Myoglobulin or Uracil) by eluting from a size-exclusion column (e.g., aTSK Guard SWXL column) in fractions collected at 10 minutes to 11minutes or 11 minutes to 12 minutes after the beginning of fractioncollection when said sample is run on an HPLC at a flow rate of 0.5mL/min. In specific embodiments, the placenta-derived adherent cellexosomes described herein elute in the first detectable peak of a samplecollected from a size-exclusion column (e.g., a TSK Guard SWXL column)after said sample is run on an HPLC at a flow rate of 0.5 mL/min.

The isolation of exosomes can be based on size, e.g., a diameter ofabout 50-150 nm. Exosome size, therefore, allows for the use oftechniques such as high-performance liquid chromatography (HPLC)combined with size-exclusion chromatography to isolate substantiallypure populations of exosomes and/or to determine the purity of isolatedexosome populations. In particular, exosomes purified and/or isolated byone of the methods described herein can be subsequently purified by HPLCto remove contaminating macromolecules and other undesired contaminants.Methods of using HPLC and size exclusion chromatography to separatemixed populations of molecules are well-known in the art and rely uponsemiporous substrates and ion/pH gradients to differentially separatethe various components of mixed populations.

In one embodiment, the placenta-derived adherent cell exosomes describedherein (e.g., exosomes previously isolated from culture medium) arefurther purified using a method comprising HPLC (e.g., using an Agilent1200 Series LC system). In a particular embodiment, said method furthercomprises using a size-exclusion chromatography column (e.g., TSK GuardSWXL and/or TSK gel G4000, Tosoh Corp.) and a buffer (e.g., 20 mM K₂PO₄,150 mM NaCl, pH 7.2) at a fixed flow rate (e.g., 0.5 ml/min). In aspecific embodiment, the placenta-derived adherent cell exosomesdescribed herein have a diameter of 50-150 nm.

5.2.2. In Vivo Antibody Capture

In a particular embodiment, placenta-derived adherent cell exosomesdescribed herein are isolated from a subject (e.g., a mouse or a human)after the introduction of placenta-derived adherent cells orplacenta-derived adherent cell exosomes described herein. In oneembodiment, a subject (e.g., a mouse) is injected with an antibodyspecific to a protein contained in or on an exosome described herein,wherein said antibody is coupled to a second high-affinity molecule(e.g., biotin). Said subject (e.g., a mouse) is additionally injected(e.g., injected intravenously, intraperitoneally, or intramuscularly)with placenta-derived adherent cells and/or isolated placenta-derivedadherent cells described herein. After a time sufficient for saidplacenta-derived adherent cells to produce exosomes, blood from thesubject (e.g., a mouse) is isolated containing complexes comprisingplacenta-derived adherent cell exosomes and antibodies specific to anexosome protein as described herein. Said antibody-exosome complexes canbe purified by contacting a substrate (e.g., streptavidin) that isspecific to the second high-affinity molecule (e.g., biotin) coupled tosaid antibody, and the resulting antibody-exosome complexes can befurther purified according to any of the methods described herein.

5.2.3. Quantification

The exosomes isolated from the placenta-derived adherent cell culturesdescribed herein may be quantified to determine total yield. Methods ofquantifying exosome yield are known in the art and include Bradfordassay, BCA assay, spectrophotometry (e.g., using a NanoDropspectrophotometer), direct Enzyme-Linked ImmunoSorbent Assay (e.g.CD63/CD9/CD81 ExoELISA (System Biosciences)), imaging techniques such asNanoparticle Tracking Analysis (e.g., Nanosight LM10 (MalvernInstruments) or electrical impedence-based measurement such as qNano(IZON)). According to these methods, exosome yields may be determinedeither as total amount of material isolated (e.g., mass of exosomalmaterial isolated per cell per day) or as the number of individualparticles in a defined volume of culture medium (e.g. exosome particlesper mL of culture medium).

5.2.4. Yield

The placenta-derived adherent cell exosomes described herein may beisolated in accordance with the methods described herein and theiryields may be quantified. In a specific embodiment, the placenta-derivedadherent cell exosomes described herein are isolated at a concentrationof about 0.5-5.0 mg per liter of culture medium (e.g., culture mediumwith or without serum). In another specific embodiment, theplacenta-derived adherent cell exosomes described herein are isolated ata concentration of about 2-3 mg per liter of culture medium (e.g.,culture medium containing serum). In another specific embodiment, theplacenta-derived adherent cell exosomes described herein are isolated ata concentration of about 0.5-1.5 mg per liter of culture medium (e.g.,culture medium lacking serum).

5.2.5. Storage and Preservation

The placenta-derived adherent cell exosomes described herein can bepreserved, that is, placed under conditions that allow for long-termstorage, or conditions that inhibit degradation of the exosomes.

In certain embodiments, the placenta-derived adherent cell exosomesdescribed herein can be stored after collection according to a methoddescribed above in a composition comprising a buffering agent at anappropriate temperature. In certain embodiments, the placenta-derivedadherent cell exosomes described herein are stored frozen, e.g., atabout −20° C. or about −80° C.

In certain embodiments, the placenta-derived adherent cell exosomesdescribed herein can be cryopreserved, e.g., in small containers, e.g.,ampoules (for example, 2 mL vials). In certain embodiments, theplacenta-derived adherent cell exosomes described herein arecryopreserved at a concentration of about 0.1 mg/mL to about 10 mg/mL.

In certain embodiments, the placenta-derived adherent cell exosomesdescribed herein are cryopreserved at a temperature from about −80° C.to about −180° C. Cryopreserved exosomes can be transferred to liquidnitrogen prior to thawing for use. In some embodiments, for example,once the ampoules have reached about −90° C., they are transferred to aliquid nitrogen storage area. Cryopreservation can also be done using acontrolled-rate freezer. Cryopreserved exosomes can be thawed at atemperature of about 25° C. to about 40° C. before use.

In certain embodiments, the placenta-derived adherent cell exosomesdescribed herein are stored at temperatures of about 4° C. to about 20°C. for short periods of time (e.g., less than two weeks).

5.3. Compositions

Further provided herein are compositions, e.g., pharmaceuticalcompositions, comprising the placenta-derived adherent cell exosomesprovided herein. See, e.g., Section 5.1. The compositions describedherein are useful in the treatment of certain diseases and disorders insubjects (e.g., human subjects) wherein treatment with exosomes isbeneficial. See Section 5.4.1.

In certain embodiments, in addition to comprising the placenta-derivedadherent cell exosomes provided herein, the compositions (e.g.,pharmaceutical compositions) described herein comprise apharmaceutically acceptable carrier. As used herein, the term“pharmaceutically acceptable” means approved by a regulatory agency ofthe Federal or a state government or listed in the U.S. Pharmacopeia orother generally recognized pharmacopeiae for use in animals, and moreparticularly in humans. The term “carrier,” as used herein in thecontext of a pharmaceutically acceptable carrier, refers to a diluent,adjuvant, excipient, or vehicle with which the pharmaceuticalcomposition is administered. Saline solutions and aqueous dextrose andglycerol solutions can also be employed as liquid carriers, particularlyfor injectable solutions. Suitable excipients include starch, glucose,lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodiumstearate, glycerol monostearate, talc, sodium chloride, dried skim milk,glycerol, propylene, glycol, water, ethanol and the like. Examples ofsuitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by JP Remington and AR Gennaro, 1990, 18^(th)Edition.

In certain embodiments, the compositions described herein additionallycomprise one or more buffers, e.g., saline, phosphate buffered saline(PBS), Dulbecco's PBS (DPBS), and/or sucrose phosphate glutamate buffer.In other embodiments, the compositions described herein do not comprisebuffers. In certain embodiments, the compositions described hereinadditionally comprise plasmalyte.

In certain embodiments, the compositions described herein additionallycomprise one or more salts, e.g., sodium chloride, calcium chloride,sodium phosphate, monosodium glutamate, and aluminum salts (e.g.,aluminum hydroxide, aluminum phosphate, alum (potassium aluminumsulfate), or a mixture of such aluminum salts). In other embodiments,the compositions described herein do not comprise salts.

The compositions described herein can be included in a container, pack,or dispenser together with instructions for administration.

The compositions described herein can be stored before use, e.g., thecompositions can be stored frozen (e.g., at about −20° C. or at about−80° C.); stored in refrigerated conditions (e.g., at about 4° C.); orstored at room temperature.

5.3.1. Formulations and Routes of Administration

The amount of placenta-derived adherent cell exosomes (see Section 5.1)or a composition described herein (see Section 5.3) which will beeffective for a therapeutic use in the treatment and/or prevention of adisease or condition will depend on the nature of the disease, and canbe determined by standard clinical techniques. The precise dosage ofplacenta-derived adherent cell exosomes, or compositions thereof, to beadministered to a subject will also depend on the route ofadministration and the seriousness of the disease or condition to betreated, and should be decided according to the judgment of thepractitioner and each subject's circumstances. For example, effectivedosages may vary depending upon means of administration, target site,physiological state of the patient (including age, body weight, andhealth), whether the patient is human or an animal, other medicationsadministered, and whether treatment is prophylactic or therapeutic.Treatment dosages are optimally titrated to optimize safety andefficacy.

Administration of the placenta-derived adherent cell exosomes describedherein (see Section 5.1), or compositions thereof (see Section 5.3) canbe done via various routes known in the art. In certain embodiments, theplacenta-derived adherent cell exosomes described herein, orcompositions thereof are administered by local, systemic, subcutaneous,parenteral, intravenous, intramuscular, topical, oral, intradermal,transdermal, or intranasal, administration. In a specific embodiment,said administration is via intravenous injection. In a specificembodiment, said administration is via subcutaneous injection. In aspecific embodiment, said administration is topical. In another specificembodiment, the placenta-derived adherent cell exosomes, or compositionsthereof, are administered in a formulation comprising an extracellularmatrix. In another specific embodiment, the placenta-derived adherentcell exosomes, or compositions thereof, are administered in combinationwith one or more additional delivery device, e.g., a stent. In anotherspecific embodiment, the placenta-derived adherent cell exosomes, orcompositions thereof, are administered locally, e.g., at or around thesite of an area to be treated with said exosomes or compositions, suchas hypoxic tissue (e.g., in treatment of ischemic diseases) or draininglymph nodes.

5.4. Methods of Use

5.4.1. Treatment of Diseases that Benefit from Angiogenesis

The placenta-derived adherent cell exosomes described herein (seeSection 5.1), and compositions thereof (see Section 5.3), promoteangiogenesis, and, therefore can be used to treat diseases and disordersthat benefit from angiogenesis. Accordingly, provided herein are methodsof using the placenta-derived adherent cell exosomes described herein,or compositions thereof, to promote angiogenesis in a subject in needthereof. As used herein, the term “treat” encompasses the cure of,remediation of, improvement of, lessening of the severity of, orreduction in the time course of, a disease, disorder or condition, orany parameter or symptom thereof in a subject. In a specific embodiment,the subject treated in accordance with the methods provided herein is amammal, e.g., a human.

In one embodiment, provided herein are methods of inducingvascularization or angiogenesis in a subject, said methods comprisingadministering to the subject the placenta-derived adherent cell exosomesprovided herein, or a composition thereof. Accordingly, the methodsprovided herein can be used to treat diseases and disorders in a subjectthat that benefit from increased angiogenesis/vascularization. Examplesof such diseases/conditions that benefit from increased angiogenesis,and therefore can be treated with the placenta-derived adherent cellexosomes and compositions described herein included, without limitation,myocardial infarction, congestive heart failure, peripheral arterydisease, critical limb ischemia, peripheral vascular disease,hypoplastic left heart syndrome, diabetic foot ulcer, venous ulcer, orarterial ulcer.

In one embodiment, provided herein are methods of treating a subjecthaving a disruption of blood flow, e.g., in the peripheral vasculature,said methods comprising administering to the subject theplacenta-derived adherent cell exosomes provided herein, or acomposition thereof. In a specific embodiment, the methods providedherein comprise treating a subject having ischemia with theplacenta-derived adherent cell exosomes provided herein, or acomposition thereof. In certain embodiments, the ischemia is peripheralarterial disease (PAD), e.g., is critical limb ischemia (CLI). Incertain other embodiments, the ischemia is peripheral vascular disease(PVD), peripheral arterial disease, ischemic vascular disease, ischemicheart disease, or ischemic renal disease.

5.4.2. Patient Populations

In certain embodiments, the placenta-derived adherent cell exosomesdescribed herein (see Section 5.1) are administered to a subject in needof therapy for any of the diseases or conditions described herein (seeSection 5.4.1). In another embodiment, a composition described herein(see Section 5.3) is administered to a subject in need of therapy forany of the diseases or conditions described herein. In certainembodiments said subject is a human.

In a specific embodiment, the placenta-derived adherent cell exosomes orcompositions described herein are administered to a subject (e.g., ahuman) in need of a therapy to increase angiogensis and/orvascularization.

5.5. Kits

Provided herein is a pharmaceutical pack or kit comprising one or morecontainers filled with one or more of the ingredients of thepharmaceutical compositions described herein, i.e., compositionscomprising the placenta-derived adherent cell exosomes described herein.Optionally associated with such container(s) can be a notice in the formprescribed by a governmental agency regulating the manufacture, use orsale of pharmaceuticals or biological products, which notice reflectsapproval by the agency of manufacture, use or sale for humanadministration.

The kits described herein can be used in the above methods (see Section5.4). The compositions described herein can be prepared in a form thatis easily administrable to an individual. For example, the compositioncan be contained within a container that is suitable for medical use.Such a container can be, for example, a sterile plastic bag, flask, jar,or other container from which the compositions can be easily dispensed.For example, the container can be a blood bag or other plastic,medically-acceptable bag suitable for the intravenous administration ofa liquid to a recipient.

5.6. Delivery Systems

The placenta-derived adherent cell exosomes described herein, andcompositions thereof, can be loaded with agent(s), e.g., pharmaceuticalagent(s), exogenous to the exosomes. Such exosomes and compositionsthereof can, for example, be taken up into target cells, e.g., cellsother than the cell type from which the exosomes were obtained, andthereby deliver the exogenous agent(s), e.g., pharmaceutical agent(s),into the target cells.

Accordingly, provided herein are methods of loading placenta-derivedadherent cell exosomes with exogenous agents, for example,pharmaceutical agents. In one embodiment, such a method comprisesincubating a placenta-derived adherent cell exosome and an exogenousagent, e.g., pharmaceutical agent, for example incubating at roomtemperature, with or without saponin permeabilization, freeze/thawcycles, sonication, or extrusion. See, e.g., Haney et al., 2015, J.Controlled Release 207:18-30, which is incorporated herein by reference.In certain embodiments, the exogenous agent, e.g., pharmaceutical agent,is loaded into the placenta-derived adherent cell exosome by incubationat room temperature. In certain embodiments, the exogenous agent, e.g.,pharmaceutical agent, is loaded into the placenta-derived adherent cellexosome by incubation at 10° C., 15° C., 18° C., 20° C., 22° C., 25° C.,28° C., 30° C., 35° C., or 40° C. In specific embodiments, incubation,e.g., incubation at room temperature, is performed without saponinpermeabilization of the placenta-derived adherent cell exosome. Inspecific embodiments, incubation, e.g., incubation at room temperature,is performed with saponin permeabilization of the placenta-derivedadherent cell exosome. In certain embodiments, the exogenous agent,e.g., pharmaceutical agent, is loaded into the placenta-derived adherentcell exosome by freeze/thaw cycles, for example, 1, 2, 3, 4, 5, or morefreeze/thaw cycles. In certain embodiments, the exogenous agent, e.g.,pharmaceutical agent, is loaded into the placenta-derived adherent cellexosome by sonication. In certain embodiments, the exogenous agent,e.g., pharmaceutical agent, is loaded into the placenta-derived adherentcell exosome by extrusion. In certain embodiments, the exogenous agent,e.g., pharmaceutical agent, is loaded into the placenta-derived adherentcell exosome by electroporation. In specific embodiments, methods ofloading exosomes with exogenous agents, for example, pharmaceuticalagents, comprise any combination of the above.

In certain embodiments, the exogenous agent, e.g., pharmaceutical agent,incorporated into the placenta-derived adherent cell placenta-derivedadherent cell exosomes described herein comprises a polypeptide. Inspecific embodiments, the exogenous agent, e.g., pharmaceutical agent isa binding agent, for example, an antibody, such as, e.g., a human,humanized, or chimeric antibody, or antigen-binding fragment thereof. Inparticular embodiments, the antibody is a monospecific, bispecific ormultispecific antibody, or antigen-binding fragment thereof. In yetother particular embodiments, the antibody or antigen binding fragmentthereof is a single-chain antibody or a Fab fragment. In specificembodiments, the antibody or antigen-binding fragment thereof would notinternalize into a target cell without the aid of the placenta-derivedadherent cell exosome.

In certain embodiments, the exogenous agent, e.g., pharmaceutical agent,loaded into the placenta-derived adherent cell exosomes described hereincomprises one or more nucleic acids. In specific embodiments, the one ormore nucleic acids comprise a siRNA or an miRNA.

In certain embodiments, the exogenous agent, e.g., pharmaceutical agent,loaded into the placenta-derived adherent cell exosomes described hereincomprises one or more gene-modifying components. In specificembodiments, the one or more gene-modifying components comprise aCRISPR-Cas system. In more specific embodiments, the CRISPR-Cas systemcomprises a guide RNA. In more specific embodiments, the CRISPR-Cassystem comprises an endonuclease. In more specific embodiments, theCRISPR-Cas system comprises a guide RNA and an endonuclease. In morespecific embodiments, the CRISPR-Cas system comprises Cas9. In morespecific embodiments, the CRISPR-Cas system comprises Cpf1. In specificembodiments, the one or more gene-modifying components comprise a zincfinger nuclease. In specific embodiments, the one or more gene-modifyingcomponents comprise a transcription activator-like effector nuclease(TALEN) system.

In another embodiment, provided herein are methods of delivering anexogenous agent, e.g., a pharmaceutical agent, to a target cell, whereinthe exogenous agent is loaded into a placenta-derived adherent cellexosome. In certain embodiments, the target cell is a cell other thanthe cell type from which the exosome was obtained. In certainembodiments, the exogenous agent, e.g., pharmaceutical agent, loadedinto the placenta-derived adherent cell exosomes described hereincomprises a polypeptide. In specific embodiments, the exogenous agent,e.g., pharmaceutical agent is a binding agent, for example, an antibody,such as, e.g., a human, humanized, or chimeric antibody, orantigen-binding fragment thereof. In particular embodiments, theantibody is a monospecific, bispecific or multispecific antibody, orantigen-binding fragment thereof. In yet other particular embodiments,the antibody or antigen binding fragment thereof is a single-chainantibody or a Fab fragment. In specific embodiments, the antibody orantigen-binding fragment thereof would not internalize into a targetcell, e.g., a cell other than the cell type from which the exosomes wereobtained, without the aid of the placenta-derived adherent cell exosome.In certain embodiments, the exogenous agent, e.g., pharmaceutical agent,loaded into the placenta-derived adherent cell exosomes described hereincomprises one or more nucleic acids. In specific embodiments, the one ormore nucleic acids comprise a siRNA. In certain embodiments, theexogenous agent, e.g., pharmaceutical agent, loaded into theplacenta-derived adherent cell exosomes described herein comprises oneor more gene-modifying components. In specific embodiments, the one ormore gene-modifying components comprise a CRISPR-Cas system. In morespecific embodiments, the CRISPR-Cas system comprises a guide RNA. Inmore specific embodiments, the CRISPR-Cas system comprises anendonuclease. In more specific embodiments, the CRISPR-Cas systemcomprises a guide RNA and an endonuclease. In more specific embodiments,the CRISPR-Cas system comprises Cas9. In more specific embodiments, theCRISPR-Cas system comprises Cpf1. In specific embodiments, the one ormore gene-modifying components comprise a zinc finger nuclease. Inspecific embodiments, the one or more gene-modifying components comprisea transcription activator-like effector nuclease (TALEN) system.

Provided herein are methods of administering to an individualplacenta-derived adherent cell exosomes comprising exogenous agents, forexample, pharmaceutical agents, to a subject, e.g., a human. In certainembodiments, the exogenous agent, e.g., pharmaceutical agent, comprisesa polypeptide. In specific embodiments, the exogenous agent, e.g.,pharmaceutical agent, is a binding agent, for example, an antibody, suchas, e.g., a human, humanized, or chimeric antibody, or antigen-bindingfragment thereof. In particular embodiments, the antibody is amonospecific, bispecific or multispecific antibody, or antigen-bindingfragment thereof. In yet other particular embodiments, the antibody orantigen binding fragment thereof is a single-chain antibody or a Fabfragment. In specific embodiments, the antibody or antigen-bindingfragment thereof would not internalize into a target cell without theaid of the placenta-derived adherent cell exosome. In certainembodiments, the exogenous agent, e.g., pharmaceutical agent, loadedinto the placenta-derived adherent cell exosomes described hereincomprises one or more nucleic acids. In specific embodiments, the one ormore nucleic acids comprise an siRNA or an miRNA. In certainembodiments, the exogenous agent, e.g., pharmaceutical agent, loadedinto the placenta-derived adherent cell exosomes described hereincomprises one or more gene-modifying components. In specificembodiments, the one or more gene-modifying components comprise aCRISPR-Cas system. In more specific embodiments, the CRISPR-Cas systemcomprises a guide RNA. In more specific embodiments, the CRISPR-Cassystem comprises an endonuclease. In more specific embodiments, theCRISPR-Cas system comprises a guide RNA and an endonuclease. In morespecific embodiments, the CRISPR-Cas system comprises Cas9. In morespecific embodiments, the CRISPR-Cas system comprises Cpf1. In specificembodiments, the one or more gene-modifying components comprise a zincfinger nuclease. In specific embodiments, the one or more gene-modifyingcomponents comprise a transcription activator-like effector nuclease(TALEN) system.

Also provided herein are compositions comprising placenta-derivedadherent cell exosomes loaded with one or more exogenous agents, forexample, pharmaceutical agents. In certain embodiments, the exogenousagent, e.g., pharmaceutical agent, incorporated into theplacenta-derived adherent cell exosomes described herein comprises apolypeptide. In specific embodiments, the exogenous agent, e.g.,pharmaceutical agent is a binding agent, for example, an antibody, suchas, e.g., a human, humanized, or chimeric antibody, or antigen-bindingfragment thereof. In particular embodiments, the antibody is amonospecific, bispecific or multispecific antibody, or antigen-bindingfragment thereof. In yet other particular embodiments, the antibody orantigen binding fragment thereof is a single-chain antibody or a Fabfragment. In specific embodiments, the antibody or antigen-bindingfragment thereof would not internalize into a target cell without theaid of the placenta-derived adherent cell exosome. In certainembodiments, the exogenous agent, e.g., pharmaceutical agent, loadedinto the placenta-derived adherent cell exosomes described hereincomprises one or more nucleic acids. In specific embodiments, the one ormore nucleic acids comprise an siRNA or an miRNA. In certainembodiments, the exogenous agent, e.g., pharmaceutical agent, loadedinto the placenta-derived adherent cell exosomes described hereincomprises one or more gene-modifying components. In specificembodiments, the one or more gene-modifying components comprise aCRISPR-Cas system. In more specific embodiments, the CRISPR-Cas systemcomprises a guide RNA. In more specific embodiments, the CRISPR-Cassystem comprises an endonuclease. In more specific embodiments, theCRISPR-Cas system comprises a guide RNA and an endonuclease. In morespecific embodiments, the CRISPR-Cas system comprises Cas9. In morespecific embodiments, the CRISPR-Cas system comprises Cpf1. In specificembodiments, the one or more gene-modifying components comprise a zincfinger nuclease. In specific embodiments, the one or more gene-modifyingcomponents comprise a transcription activator-like effector nuclease(TALEN) system.

6. EXAMPLES

6.1. Example 1: Methods of Isolating Exosomes

Placenta-derived adherent cell exosomes were isolated from CD10⁺,CD200⁺, CD105⁺, CD34⁻ placenta-derived adherent cell cultures accordingto the following methods:

Fetal bovine serum (FBS) was depleted of contaminating bovine exosomesby ultracentrifugation at 10,000×g for 1 hour, then at 100,000×g for 18hours at 4° C. The resulting supernatant was filtered through a 0.2 μmfilter prior to storage and use. Exosome-depleted FBS was mixed withDulbecco's Modified Eagle's Medium (DMEM) for 24 hours to produce growthmedium. Growth medium was warmed to 37° C. and placenta-derived adherentcells were plated at a density of 300-1,000 cells/cm² and cultured for 3days or until 80% confluent in a cell culture platform device (CorningCELLSTACK 10-Tray with CELLBIND coating). Placenta-derived adherent cellexosomes were plated at passage six, and culture supernatant wascollected at passage seven. The placental source of the placenta-derivedadherent cells used to isolate the exosomes described herein did notinclude chorionic villi, thus the placenta-derived adherent cells usedto produce placenta-derived adherent cell exosomes did not includechorionic villi mesenchymal stem cells.

After the removal of culture supernatant, the cell culture monolayer waswashed with PBS and replenished with serum-free medium (DMEM and 1×glutamine). After 48 hours, the serum-free culture supernatant wascollected and subjected to exosome enrichment and isolation procedure asdescribed below. The resulting cell monolayer was harvested for cellcount, exosome yield comparison and comparison studies.

Supernatant from confluent serum-containing and serum-free cultures wascollected and centrifuged in 500 mL tubes at 400×g for 5-15 minutes at4° C. Centrifuged supernatant was then transferred to new 500 mL tubesand centrifuged at 3000×g for 20-60 minutes at 4° C. Supernatant wasthen transferred to appropriate ultracentrifuge tubes and centrifuged at100,000×g for 60 minutes at 4° C. A small aliquot of the resultingexosome-depleted supernatant was saved for further analysis, and theremainder was discarded. The pellet, containing exosomes, wasresuspended in PBS or injectable saline and ultracentrifugation wasrepeated. The supernatant was discarded and the resulting pellet wasre-suspended in PBS or injectible saline, and analyzed according to theexamples described below. The resuspended pellets are referred to as“Growth Exosomes” when isolated from cultures containing FBS, or“Serum-Free Exosomes” when isolated from cultures lacking FBS.

The isolated exosome compositions were aliquoted in 2 mL cryo vials andstored at −20 to −80° C.

6.2. Example 2: Confirmation of Placenta-Derived Adherent Cell Exosomes.

The results presented herein demonstrate that the non-cellular materialsisolated from cell culture supernatants in Example 1 are exosomes.

6.2.1. Detection Using Exosomal Marker Antibody Array

Placenta-derived adherent cell exosomes were isolated as in Section 6.1.Four hundred micrograms of isolated material was analyzed usingExo-Check™ exosome antibody array (System Biosciences) according to themanufacturer's protocol. Exosome lysates were incubated overnight on theantibody array and the sample was imaged using a Kodak Gel-Logic systemand developed after a 2 minute luminescence exposure. As shown in FIG. 1(bottom) placenta-derived adherent cell exosome material resulted indetectable signal for the four markers FLOT-1, ALIX, ANXA5 and TSG101.The use of a positive control sample, included as part of the array kit,resulted in a detectable signal for these markers as well (FIG. 1 top).

6.2.2. Shape and Size Characteristics of Placenta-Derived Adherent CellExosome Samples

Isolated exosomes were negative stained and imaged using transmissionelectron microscopy (TEM) (FIG. 2A) and were found to exist as discretebodies ˜100 nm in diameter, often in a cup-like shape (arrows), bothcharacteristics of exosomes. The purified exosomes were further analyzedby nanoparticle tracking analysis (NTA) to determine the sizedistribution of the isolated population. As shown in FIG. 2B,placenta-derived adherent cell exosomes were an average size of 139 nm,consistent with the known size of exosomes isolated from other celltypes.

The morphological characteristics and identified protein markersdemonstrated that the material isolated from placenta-derived adherentcells using the above methods are exosomes.

6.3. Example 3: Purification of Placenta-Derived Adherent Cell Exosomes

6.3.1. Exosome Purification by Size-Exclusion High-Performance LiquidChromatography

Placenta-derived adherent cell exosomes were isolated as in Section 6.1and further analyzed by size-exclusion high-performance liquidchromatography (SE-HPLC). Elution profiles of exosomes from chorionicvilli mesenchymal stem cells (MSCs) were compared to exosomes obtainedfrom serum-containing (“Growth Supernatant”) and serum-free (“Serum FreeSupernatant) placenta-derived adherent cell cultures. Elution profilesof exosome preparations (FIGS. 3B-D, arrows) were analyzed in comparisonto the elution peaks of known culture medium contaminants (FIG. 3A).

Chorionic villi MSC exosomes contained detectable levels of thecontaminants Thyroglobulin and BSA (FIG. 3B). Growth Supernatantplacenta-derived adherent cell exosomes also contained detectable levelsof Thyroglobulin and BSA (FIG. 3C), while Serum Free Supernatantplacenta-derived adherent cell exosomes contained no detectable levelsof any of such contaminants (FIG. 3D). Elution profiles of exosomesisolated from either Growth Supernatant or Serum Free cultures werecharacterized by a peak consistent with that of the positive controlexosomes. In the case of Serum Free culture exosomes, the elutionprofile was present as a single species in the predicted exosome elutionfraction (FIG. 3D). These results further demonstrate that the isolatedplacenta-derived adherent cell microvesicles are exosomes, and thatexosomes isolated from cells grown in serum-free conditions lack typicalcontaminants as compared to exosomes prepared from cells grown in thepresence of FBS.

6.4. Example 4: Placenta-Derived Adherent Cell Exosome Molecular Markers

In the present study, FACS was used to examine which of a panel of cellsurface markers are contained on the surface of exosomes, and whichsurface markers are unique to placenta-derived adherent cell exosomes ascompared to exosomes derived from other cell types.

In addition to surface marker proteins, it is known that exosomes maycontain small nucleic acids, including miRNAs. The composition of miRNAscontained within exosomes may vary between cell types from whichexosomes were derived, and the presence and abundance of miRNAs can bedetermined by methods known in the art, including real-time PCR(RT-PCR). In this study, miRNA content of placenta-derived adherent cellexosomes was examined and compared to exosomes derived from chorionicvilli mesenchymal stem cells.

6.4.1. Placenta-Derived Adherent Cell Exosome Surface Markers

Placenta-derived adherent cell exosomes isolated as in Section 6.1 wereanalyzed by FACS, and the composition of surface markers was compared toexosomes isolated from either chorionic villi MSCs or pre-adipocyteMSCs. The exosomes from these three cell types were determined todifferentially contain several surface markers (Table 1). Using FACSanalysis, exosomes from placenta-derived adherent cells were found tocontain a number of markers unique or enriched in these exosomes ascompared to exosomes from chorionic villi MSCs or pre-adipocyte MSCs. Ascompared to exosomes isolated from chorionic villi MSCs or pre-adipocyteMSCs, placenta-derived adherent cell exosomes contained CD10 and CD55,both of which were absent in both mesenchymal stem cell exosomepopulations. Placenta-derived adherent cell exosomes also contained ahigher level of CD49c, CD82, CD90, and CD142, e.g., a 0.5 to 1 loghigher level, as compared to the two mesenchymal stem cell exosomepopulations (Table 1). Conversely, the placenta-derived adherent cellexosomes contained a lower level of CD164, e.g., a 0.5 to 1 log lowerlevel, as compared to the two mesenchymal stem cell exosome populations(Table 1). Placenta-derived mesenchymal stem cell exosomes alsocontained detectable levels of CD295, which was not detected onpre-adipocyte MSC exosomes (not shown). As shown in Table 1, the numberof “+” symbols in each entry is correlated with the level of theindicated markers, whereas “−” symbols represent no detectable levels ofthe indicated marker.

TABLE 1 Surface antigens of placenta-derived adherent cell exosomescompared to pre-adipocyte MSC exosomes and chorionic villi MSC exosomesPlacenta-Derived Adherent Cell Pre-Adipocyte MSC Placental MSC AntigenExosomes Exosome Exosome CD9 ++ ++ ++ CD10 + − − CD13 ++ ++ ++ CD29 ++++ ++ CD44 + + ++ CD49b + +/− + CD49c ++ − + CD55 + − − CD59 ++ ++ ++CD63 ++ ++ ++ CD73 ++ + ++ CD81 +++ +++ +++ CD82 ++ + + CD90 ++ + +CD98 + + + CD105 + +/− + CD141 + + − CD142 ++ + + CD151 ++ ++ ++ CD164 +++ ++ CD200 + + +

6.4.2. Placenta-Derived Adherent Cell Exosome miRNA Markers

6.4.2.1. Placenta-Derived Adherent Cell Exosomes and Chorionic VilliMesenchymal Stem Cell Exosomes Differ in their miRNA Content

Placenta-derived adherent cell exosomes were isolated using theExoquick™ precipitation method according to the manufacturer's protocol(System Biosciences). Culture supernatant was centrifuged for 15 minutesat 3000×g, and the resulting pellet was discarded. Two milliliters ofthe resulting supernatant was added to 10 ml tissue culture supernatantand mixed by inversion. The resulting mixture was incubated at 4° C.overnight. After incubation, the mixture was centrifuged at 1500×g for30 minutes, the supernatant was discarded and the pellet was isolated.The remaining liquid was removed by aspiration after centrifuging foradditional 5 minutes at 1500×g. The pellet was reconstituted in 200 ulPBS.

Isolated placenta-derived adherent cell exosomes and chorionic villi MSCexosomes (Salomon et al., 2013, PLOS ONE, 8:7, e68451) were lysed andRNA was isolated using Ambion's mirVana miRNA isolation kit (Cat#1566)according to the manufacturer's instructions. In brief, exosomes werelysed with Lysis/Binding Buffer (supplemented with miRNA HomogenateAdditive) on ice for 10 minutes, RNA was isolated usingacid—phenol:chloroform extraction, and precipitated with ethanol washes.Fifty nanograms of purified RNA was reverse transcribed and probe setsfor 17 miRNAs and the control RNA RNU44 were used to amplify the samplesaccording to the TagMan® qPCR system (Life Technologies). Ct values werecalculated for each sample.

As shown in FIG. 4A, placenta-derived adherent cell exosomes containhigher levels of all miRNAs analyzed in each of the three groups ascompared to chorionic villi MSCs. These data further indicate that theplacenta-derived adherent cell exosomes described herein are distinctfrom chorionic villi mesenchymal stem cell exosomes on the basis ofmiRNA content. miRNAs miR-451, miR-142-3p, miR-16-5p, miR-296-5p,miR-233-3p, miR-92, Let-7d*, miR-422a, and miR-133b all showed adifference of Ct values greater than 2 for chorionic villi mesenchymalcell exosomes as compared to placenta-derived adherent cell exosomes(see Table 2), indicating significantly higher presence of these miRNAsin placenta-derived adherent cell exosomes. Data shown are from oneexperiment performed in triplicate.

TABLE 2 Ct values of miRNAs from placenta-derived adherent cell exosomesand chorionic villi mesenchymal stem cell exosomes. Chorionic VilliPlacenta-Derived Mesenchymal Stem miRNA Name Adherent Cell Ct Cell CtDifference miR-451 26.906 37.331 10.425 miR-142-3p 30.405 38.031 7.626miR-16-5p 20.264 26.87 6.606 miR-296-5p 25.017 30.786 5.769 miR-223-3p27.951 33.437 5.486 miR-92 19.689 24.958 5.269 Let-7d* 25.905 30.6584.753 miR-422a 29.413 31.999 2.586 miR-133b 31.443 33.806 2.363

6.4.3. Placenta-Derived Adherent Cell Exosomes Contain AngiogenicProteins

To profile the contents of placental exosomes for angiogenic secretedcytokines, an Angioplex was performed using Human Mag Bead kit,HAGP1MAG-12K (Millipore). This kit contains analytes for detecting thefollowing 17 cytokines: Leptin, Endoglin, Follistatin, HGF,Angiopoietin-2, G-CSF, FGF-1, FGF-2, VEGF-A, VEGF-C, VEGF-D, EGF,Endothelin-1, BMP-9, IL-8, HB-EGF, and PLGF. Exosomes were analyzedaccording to the manufacturer's suggested protocol.

Growth Exosomes and Serum Free Exosomes were isolated as described inExample 1. The exosomes were prepared using a 100 μg proteinconcentration preparation in PBS, which was diluted 10.8× forangiogenesis analyte detection profiling. Exosomes were sonicated ineither a water-bath to disperse the exosomes for surface markeranalysis, or using a probe to lyse the exosomes for intra-exosomalanalysis. As a comparison, growth medium cleared of cells(“pre-enrichment conditioned”) and growth medium ultracentrifuged toremove exosomes and other debris (“exosome depleted”) were analyzedusing the same method.

Placenta-derived adherent cell exosomes displayed detectable levels ofseveral cytokines including HGF and VEGF-A (FIG. 4B), Endoglin andLeptin (FIG. 4C), Angiopoietin-2, G-CSF, Follistatin, FGF-2, and IL-8(not shown). Nearly all of these cytokines were present at a lower levelin all growth media tested, indicating that the cytokine levels arespecifically associated with the exosomes. These data indicate thatplacenta-derived adherent cell exosomes contain protein factors that areknown to promote angiogenesis.

6.5. Example 5: Placenta-Derived Adherent Cell Exosome Labeling andCellular Uptake

To determine if isolated placenta-derived adherent cell exosomes arecapable of delivering their contents to cultured cells, exosomal uptakeassays were performed using blood obtained in TruCulture tubes. Isolatedplacenta-derived adherent cell exosomes (see Section 6.1) were labeledwith 2 μM of the fluorophore PKH26 by adding 2 μl PKH26 to 1 ml exosomesuspension and incubated for 5 minutes at room temperature in the dark.Exosomes were recovered using a Centricon 10 kDa molecular weight cutoffcolumn according to the manufacturer's instructions. Labeled exosomeswere added to 200 μl TruCulture blood preparations and incubated for 24hours at 37° C. in 5% CO₂. After incubation, 100 μl blood was stainedwith leukocyte lineage specific antibodies for 15 minutes at roomtemperature in the dark. After incubation, red blood cells were lysedwith ACK lysing solution and washed 3× with PBS, 1% FBS. Exosome uptakeon particular leukocyte populations were determined by lineage specificgating in FlowJo software and PKH26 fluorescence. As shown in FIG. 5,several lineage types stained positive for PKH26, indicating thatplacenta-derived adherent cell exosomes can be taken up by culturedcells.

6.6. Example 6: Placenta-Derived Adherent Cell Exosomes Enhance theProliferation of Human Vascular Endothelial Cells

Human Vascular Endothelial Cells (HUVECs) were plated at a concentrationof 14,000 cells/well on matrigel and incubated with either increasingamounts of placenta-derived adherent cell exosomes in the absence ofserum (see Section 6.1), 3×10⁴ placenta-derived adherent cells in theabsence of serum, or culture medium alone. As shown in FIG. 6, theproliferation of HUVECs correlated with the concentration ofplacenta-derived adherent cell exosomes added to the culture. These dataindicate that placenta-derived adherent cell exosomes can enhance theproliferation of cultured human cells.

6.7. Example 7: Exosome-Mediated Effects on Cytokine Production in WholeBlood

6.7.1. Experimental Design

An in vitro model for cytokine expression in the blood as a result ofimmunogenic stimulation was used. In culture tubes, 2 ml of Myriad RBMTruCulture™ media was incubated with 1 ml of whole human blood andincreasing amounts of either placenta-derived adherent cell exosomes (0,6, 12, 24, 47 and 94n/ml) or placenta-derived adherent cells (2.5, 5,10, 20 or 40×10³ cells) for 1 hour before stimulation with 10 ng/mllipopolysaccharide (LPS) for 24 hours. Cytokines produced in thecultured blood were detected using HCYTOMAG-60K|MILLIPLEX MAP HumanCytokine/Chemokine Magnetic Bead Panel—Immunology Multiplex Assay(Millipore)

6.7.2. Placenta-Derived Adherent Cells and Placenta-Derived AdherentCell Exosomes Alter Cytokine Production in Whole Blood

As shown in FIG. 7A, placenta-derived adherent cells andplacenta-derived adherent cell exosomes reduce the levels of specificcytokines in a dose-dependent manner. The pro-inflammatory cytokinesTNF-α, IL-12p40, MIP1b and IL-1B were all reduced when incubated withincreasing amounts of placenta-derived adherent cells orplacenta-derived adherent cell exosomes in LPS-stimulated whole blood.Conversely, MCPJ levels were increased by both placenta-derived adherentcells and placenta-derived adherent cell exosomes in stimulated wholeblood, indicating that placenta-derived adherent cells andplacenta-derived adherent cell exosomes can affect cytokine productionin cultured cells.

6.7.3. Placenta-Derived Adherent Cells and Placenta-Derived AdherentCell Exosomes Differentially Regulate a Subset of Cytokines inStimulated Blood

As shown in FIG. 7B, placenta-derived adherent cell exosomes do notenhance the levels of specific cytokines that are enhanced in thepresence of placenta-derived adherent cells. IFN-γ, IL-6, IL-8 andGM-CSF, cytokines associated with inflammation, were not enhanced in thepresence of placenta-derived adherent cell exosomes in stimulated wholeblood. This finding indicates that placenta-derived adherent cellexosomes exert a differential effect on cultured blood than that ofcells from which they were derived.

6.8. Example 8: Placenta-Derived Adherent Cell Exosomes PromoteAngiogenesis in Vitro

Human vascular endothelial cells (HUVECs) at passage 3 or 4 were platedin 48-well microtiter dishes at a density of 28,000 cells per well.Cells were maintained in 0.5 mL of either PBD or EBM-2 culture medium(Lonza) on GRF-Matrigel for 3 days. HUVECs were then incubated withdifferent concentrations of placenta-derived adherent cell exosomesisolated from either serum-containing (“growth exosomes”) or serum-free(“SF exosomes”) placenta-derived adherent cell cultures. HUVEC growthpatterns, including node formation and tube segment length (surrogatesfor angiogenic potential) were monitored every two hours for 18-19 hourstotal. HUVEC growth parameters were determined using IncuCyte live cellimaging system (Essen BioScience) and quantified using ImageJ.

6.8.1. Growth Exosomes and Serum Free Exosomes Promote HUVEC TubeFormation

As shown in FIG. 8A, growth exosomes increased tube segment length andbranch point frequency in cultured HUVECs in a dose-dependent manner. Ascompared to HUVECs incubated with PBS, isolated purified exosomes inconcentrations ranging from 5 μg/well to 100 μg/well increased tubesegment length (8A, left). Similarly, the HUVECs treated with growthexosomes had a higher number of nodes as compared to HUVECs treated withPBS (8A, right).

Similar to the results observed with the growth exosomes,placenta-derived adherent cell exosomes isolated from serum-freecultures increased HUVEC tube segment length and branch point frequency.As shown in FIG. 8B, HUVECs cultured in the presence of exosomes rangingin concentration of 5 μg/well to 100 μg/well increased tube segmentlength (8B, left) and branch point frequency (8B, right) as compared toHUVECs cultured in the presence of PBS. These results indicate thatisolated placenta-derived adherent cell exosomes can promote vascularcell proliferation and vascular tube branching frequency in vitro,indicating a role for placenta-derived adherent cell exosomes aspro-angiogenic factors.

6.8.2. Placenta-Derived Adherent Cell Exosomes Alter Gene Expression inHUVEC Cells

Passage 3 HUVEC cells (Lonza Lot 8F3265; 032514AR) were expanded topassage 4 in complete EGM-2 Media. The cells were harvested, seeded onfibronectin coated plates at high density, and allow to attach for 3hours until ˜100% confluent. Cells were cultured in the absence of serum˜2 hours in EBM-2 medium. Cultured HUVECs were incubated withplacenta-derived adherent cell culture supernatant, 50 μg ofplacenta-derived adherent cell exosomes, or culture medium as a control.HUVEC supernatant and cell lysates were collected at 4, 24, and 48hours, and gene expression was analyzed at these early, intermediate,and late time points.

RNA was isolated using Qiagen's Rneasy Mini Kit (Cat#74134) as permanufacture's recommended protocol. 3.5 ug of RNA wasreverse-transcribed using VILO RT MM and 40 ng of cDNA was analyzed byRT-PCR using Taqman Fast Universal PCR Master Mix and Taqman Human VEGFPathway Array 96-well Fast Plates.

The results of the VEGF pathway array are shown in FIG. 8C (darkershades indicate higher expression level). Notably, HUVECs failed toexpress ACTA1, BAD, CASP9, MAP2K1, PLCG1, and SHC1 under any conditions,but 26/44 VEGF Pathway genes were up-regulated in HUVECs treated withplacenta-derived adherent cell exosomes and/or placenta-derived adherentcell culture supernatant (FIG. 8C). Importantly, many of these genes areknown to play an important role in angiogenesis.

Several gene expression subclasses were altered by placenta-derivedadherent cell exosomes and/or placenta-derived adherent cell culturesupernatants as compared to control-treated HUVECs including genesrelated to (i) cell motility (FIG. 8D), (ii) cell proliferation (FIG.8E-F), and (iii) nitric oxide production, cell permeability, and cellsurvival (FIG. 8G). These results indicate that placenta-derivedadherent cell exosomes are capable of altering the gene expressionprogram of vascular cells, e.g., towards a proliferative state. Theseresults also demonstrate that the effects of exosomes can be distinctfrom those induced by placenta-derived adherent cell culturesupernatant.

6.8.3. Example 9: Placenta-Derived Adherent Cell Exosomes Alter CytokineProduction in Macrophages

Isolated monocytes were cultured in the presence of GM-CSF for 7-10 daysuntil differentiated into macrophages. 5×10⁴ macrophages wereco-cultured with placenta-derived adherent cell exosomes orplacenta-derived adherent cells and 100 ng/mL LPS for 22-24 hrs.Secreted cytokines were profiled using a human immune multiplex panel(Millipore, HCYTO-MAG-60K). Assays were performed according tomanufacturer's protocol using a Luminex FlexMAP-3D system.

As shown in FIG. 9A, placenta-derived adherent cells andplacenta-derived adherent cell exosomes altered the levels of specificcytokines produced by monocyte-derived macrophages in a dose-dependentmanner. Specifically, TNF-α and MCP-1 were both reduced when incubatedwith increasing amounts placenta-derived adherent cell exosomes in thepresence of LPS-stimulated macrophages. Placenta-derived adherent cellsdid not reduce the levels of MCP-1 in macrophage cultures.

As shown in FIG. 9B (left, center), placenta-derived adherent cellexosomes and placenta-derived adherent cells both suppressed TNF-αsecretion in cultured macrophages, while placenta-derived adherent cellexosomes but not placenta-derived adherent cells broadly suppressedcytokine secretion (e.g., IL-8 secretion) in cultured macrophages.Notably, this is in contrast to the changes in cytokine expressionobserved in whole blood, (see Example 7). These results indicate thatplacenta-derived adherent cell exosomes can alter cytokine expression incultured immune cells in a manner distinct from the cells from which theexosomes were derived. When monocyte-derived macrophages are incubatedwith lysed placenta-derived adherent cell exosomes, IL-8 suppression isattenuated when compared to cultures incubated with intactplacenta-derived adherent cell exosomes (FIG. 9B, right). These resultsindicate that the exosome effect observed is due to the presence of theexosomes themselves, not merely to the mixture of components that makeup the exosomes.

EQUIVALENTS

The present disclosure is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of thesubject matter provided herein, in addition to those described, willbecome apparent to those skilled in the art from the foregoingdescription and accompanying figures. Such modifications are intended tofall within the scope of the appended claims.

Various publications, patents and patent applications are cited herein,the disclosures of which are incorporated by reference in theirentireties.

What is claimed:
 1. A composition comprising exosomes derived from humanplacenta-derived adherent cells, wherein said exosomes are CD9⁺, CD10⁺,CD13⁺, CD29⁺, CD44⁺, CD49b⁺, CD49c⁺, CD55⁺, CD59⁺, CD63⁺, CD73⁺, CD81⁺,CD82⁺, CD90⁺, CD98⁺, CD105⁺, CD141⁺, CD142⁺, CD151⁺, CD164⁺, CD295⁺, orCD200′.
 2. The composition of claim 1, wherein said exosomes are CD9⁺,CD10⁺, CD13⁺, CD29⁺, CD44⁺, CD49b⁺, CD49c⁺, CD55⁺, CD59⁺, CD63⁺, CD73⁺,CD81⁺, CD82⁺, CD90⁺, CD98⁺, CD105⁺, CD141⁺, CD142⁺, CD151⁺, CD164⁺,CD295⁺, and CD200′.
 3. The composition of claim 1 or 2, wherein saidexosomes are CD3−, CD11b-, CD14-, CD19-, CD33-, CD192-, HLA-A-, HLA-B-,HLA-C-, HLA-DR-, CD11c- or CD34-.
 4. The composition of any of claims1-3, wherein said exosomes are CD3-, CD11b-, CD14-, CD19-, CD33-,CD192-, HLA-A-, HLA-B-, HLA-C-, HLA-DR-, CD11c- and CD34-.
 5. Thecomposition of any one of claims 1-4, wherein said exosomes comprisenon-coding RNA molecules.
 6. The composition of claim 5, wherein saidRNA molecules are microRNAs.
 7. The composition of claim 6, wherein saidmicroRNAs are miR-218-5p, miR-133b, miR-422a, miR-564, miR-16-5p,let-7a, miR-92, miR-142-3p, miR-451, miR-124-5p, miR-223-3p, miR-630,miR-296-5p, let-7b-3p or let-7d-3p.
 8. The composition of any of claims1-7, wherein said exosomes comprise at least one marker molecule at alevel at least two-fold higher than exosomes derived from mesenchymalstem cells.
 9. The composition of any of claims 1-8, wherein saidplacenta-derived adherent cells have been passaged more than 3 times.10. The composition of any of claims 1-9, wherein said placenta-derivedadherent cells have been maintained in culture for greater than 24hours.
 11. The composition of any of claims 1-10, wherein at least 90%of said placenta-derived adherent cells are non-maternal in origin. 12.The composition of any of claims 1-11, wherein at least 99% of saidplacenta-derived adherent cells are non-maternal in origin.
 13. Thecomposition of any of claims 1-12 that is in the a form suitable forintravenous administration.
 14. A method of angiogenesis orvascularization in said subject comprising administering the compositionof any one of claims 1-13 to the subject.
 15. A method of modulating theimmune system of a said subject comprising administering the compositionof any one of claims 1-13 to the subject.
 16. A method of repairingdiseased or damages tissue in a subject comprising administering thecomposition of any one of claims 1-13 to the subject.
 17. The method ofany of claims 14-16, wherein said subject is human.
 18. A compositioncomprising exosomes derived from human placenta-derived adherent cells,wherein said exosomes are CD10⁺ and CD55⁺.
 19. A method of loadingplacenta-derived adherent cell exosomes with exogenous agents,comprising incubating a placenta-derived adherent cell exosome and anexogenous agent, such that the exogenous agent is loaded in the exosome.20. The method of claim 19, wherein the incubation is performed at roomtemperature.
 21. The method of claim 19 or 20, which comprises a step ofsaponin permeabilization.
 22. The method of claim 19 or 20, which doesnot comprise a step of saponin permeabilization.
 23. The method of claim19 or 20, which comprises one or more freeze/thaw cycles.
 24. The methodof claim 19 or 20, which comprises a step of sonication.
 25. The methodof claim 19 or 20, which comprises a step of extrusion.
 26. A method ofadministering placenta-derived adherent cell exosomes comprisingexogenous agents to an individual.
 27. The method of claim 26, whereinthe exogenous agents comprise a human, humanized, or chimeric antibody,or antigen-binding fragment thereof.
 28. The method of claim 26, whereinthe exogenous agents comprise one or more gene-modifying components. 29.The method of claim 28, wherein the gene-modifying components comprise aCRISPR-Cas system.
 30. The method of claim 29, wherein the CRISPR-Cassystem comprises a guide RNA and an endonuclease.
 31. A compositioncomprising placenta-derived adherent cell exosomes comprising one ormore exogenous agents.
 32. The composition of claim 31, wherein theexogenous agents comprise a human, humanized, or chimeric antibody, orantigen-binding fragment thereof.
 33. The composition of claim 31,wherein the exogenous agents comprise one or more gene-modifyingcomponents.
 34. The composition of claim 33, wherein the gene-modifyingcomponents comprise a CRISPR-Cas system.
 35. The composition of claim34, wherein the CRISPR-Cas system comprises a guide RNA and anendonuclease.
 36. A method of delivering an exogenous agent to a targetcell, wherein the exogenous agent is loaded into a placenta-derivedadherent cell exosome.
 37. The method of claim 36, wherein the targetcell is a cell other than the cell type from which the exosome wasobtained.
 38. The method of claim 36 or 37, wherein the exogenous agentscomprise a human, humanized, or chimeric antibody, or antigen-bindingfragment thereof.
 39. The method of claim 36 or 37, wherein theexogenous agents comprise one or more gene-modifying components.
 40. Themethod of claim 38, wherein the gene-modifying components comprise aCRISPR-Cas system.
 41. The method of claim 39, wherein the CRISPR-Cassystem comprises a guide RNA and an endonuclease.